Li ni u

Cng ngh DNA ti t hp (cn gi l k thut di truyn hay k thut gen) l mt b phn quan trng v l cng ngh cha kha (key technology) ca lnh vc cng ngh sinh hc. Cng ngh DNA ti t hp c ra i trn c s cc thnh tu ca sinh hc phn t v hin nay ang ng vai tr cch mng i vi s pht trin ca sinh hc cng nh ci to sinh gii. Cc k thut ti t hp DNA cho php cc nh cng ngh sinh hc phn lp v khuch i mt gen n t genome ca mt sinh vt c th nghin cu, bin i v chuyn n vo trong mt c th sinh vt khc. Cc k thut ny cn c gi l to dng gen do n c th sn xut ra mt s lng ln cc gen xc nh. Bn cnh cc gio trnh nh: sinh hc phn t, nhp mn cng ngh sinh hc, cng ngh t bo, cng ngh chuyn gen gio trnh cng ngh DNA ti t hp s gip sinh vin tip cn thm mt lnh vc khc ca cng ngh sinh hc thng qua vic cung cp nhng kin thc c bn theo hng to dng v biu hin gen nh sau: - Cc enzyme dng trong to dng phn t. - Cc h thng vector. - Mt s k thut c bn trong to dng gen: in di, PCR - To dng v xy dng cc th vin genomic DNA v cDNA. - Biu hin cc gen c to dng trong E. coli. Do gio trnh ny mi c xut bn ln u tin, hn na lnh vc cng ngh DNA ti t hp li rt phc tp, nn kh trnh khi thiu st hoc cha p ng c yu cu bn c. V th, chng ti rt mong nhn c nhiu kin ng gp ln xut bn sau c hon thin hn. Chng ti chn thnh cm n Qu Nng cao cht lng-D n Gio dc i hc h tr chng ti bin son gio trnh ny, PGS. TS. L Trn Bnh c bn tho v gp nhiu kin qu bu.

Cc tc gi

Ph lc

Mt s thut ng c bn
Adapter. Mt oligodeoxyribonucleotide tng hp tng t linker, nhng c mt u bng v mt u li 5 tng ng vi mt v tr ct hn ch cho php ni cDNA si i vi cac plasmid vector hoc bacteriophage vector c u tng ng (xem thm linker). Adenosine diphosphate (ADP). Mt ribonucleoside 5-diphosphate c cu to t adenine, ng ribose (5C) v hai gc phosphate. ADP c tc dng nhn phosphate trong chu trnh nng lng ca t bo. Adenosine triphosphate (ATP). Mt ribonucleoside 5-triphosphate c cu to t adenine, ng ribose (5C) v ba gc phosphate. ATP l phn t cha nng lng ha hc chnh ca t bo, chu yu c tp hp trong ty th (mitochondria) va lp th (chloroplast). Cc gc phosphate ca ATP c mang cc lin kt khi b thy phn s phng thch mt nng lng t do ln. Nng lng ca qua trinh h hp hoc quang hp c s dung tao thanh ATP t ADP. Sau o, ATP c bin i ngc tr lai thanh ADP nhiu vung khac nhau cua t bao, nng lng phong thch ra c dung iu khin cac phan ng ha sinh ni bao. i khi cng xay ra s thy phn tip ADP thanh nhng AMP (adenosine monophosphate) phng thch nng lng nhiu hn. Amino acid. L mt phn t nh mang mt gc amine (-NH3) v mt gc carboxyl (-COOH) lin kt vi cng mt nguyn t carbon. Amino acid l n v cu trc c s ca chui polypeptide. C 20 amino acid khc nhau trn cc chui polypeptide co trong t nhin. Trinh t sp xp cua cac amino acid trn chui polypeptide quyt inh cu truc va chc nng cua polypeptide va protein ma no tao thanh. Ampicillin (Amp). Cht khang sinh ban tng hp c dung trong mi trng chon lc chon cac t bao mang t bin khuyt dng hoc chon dong t bao (ti t hp) mang oan DNA c to dong. BAC (bacteria artificial chromosome). Nhim sc th nhn to ca vi khun, da trn c s plasmid F-factor, c s dng lm vector to dng. BAC c th ti bn trong E. coli vi cc on chn DNA c kch thc ln n 300 kb. Bn ct hn ch (restriction map). Trnh t cc v tr nhn bit (recognition sites) ca tt c cc enzyme hn ch (restriction enzyme hay restriction endonuclease, RE) trn mt phn t DNA.

Baz ng ng (analog base). Cht ha hoc co cu truc phn t rt ging cac base binh thng cua DNA. Chung co th thay th cac nitrogen base binh thng trong DNA va hoat ng nh mt tac nhn t bin. Trong ln sao chep tip theo cua DNA, base ng ng co th bt cp sai vi mt base binh thng, tao nn t bin im. V d: base ng ng cua adenine (A) la 2-aminopurine (AP) co th gn vao DNA vi tri ca adenine; trong ln sao chep tip o co th bt cp vi cytosine (C), trong ln sao chep tip theo na C kt cp vi guanine (G). Nh vy a din ra s thay th cp A-T bng cp G-C. Baz nit (nitrogen base). Loai phn t cu tao nn nucleic acid (DNA v RNA). Cac nitrogen base co trong nucleic acid l adenine, guanine, cytosine v thymine (DNA) hoc uracil (RNA). Trinh t sp xp cua chung doc theo phn t nucleic acid tao nn thng tin di truyn ca c th sinh vt. Bt cp b sung (complementary base pairing). S kt hp thnh tng i gia cc nitrogen base nm trn hai mch n ca chui xon kp DNA-DNA, DNARNA hoc RNA-RNA thng qua cc mi lin kt hydrogen. S bt cp mang tnh c hiu: guanine bt cp vi cytosine, cn adenine bt cp vi thymine trn DNA hoc uracil trn RNA. Bin np (transformation). L qu trnh truyn DNA ngoi lai vo mt t bo nhn, chng hn sphaeroplast hoc protoplast, v c th hp nht trong nhim sc th nh s ti t hp tng ng hoc c bin i trong mt n v sao chp t tr (autonomous replicon). S bin np c th xut hin trong cc iu kin t nhin mt s vi khun (v d: Bacillus, Haemophilus, Neisseria v Streptococcus), nhng nhiu vi khun (v d: E. coli) v cc c th sinh vt eukaryote s bin np ch c th xut hin nhng t bo thm c DNA bng cc phng php nhn to nh: ha bin np, in bin np... Bin np bng in (electroporation). K thut dng xung in to ra cc l thng tm thi trn mng sinh cht a DNA ngoi lai vo bn trong t bo vt ch. Bin tnh (denaturation). L hin tng chuyn t dng mch kp sang dng mch n ca DNA v RNA thng do nhit gy nn. Bin tnh ca protein l hin tng chuyn t cu hnh hot ng thnh dng khng hot ng. Biu hin ca gen (gene expression). L cc qu trnh phin m (transcription) v dch m (translation) ca mt gen to ra sn phm protein ca n. Cp base (base pair, bp). L lin kt A-T hoc C-G trn mt phn t DNA mch kp, v l n v o chiu di ca mt phn t DNA. Chromosome walking. K thut ny dng lp bn nhim sc th t tp hp cc on DNA ct hn ch chng ln nhau (overlapping). Bt u t mt th vin trong cha cc on DNA ni trn c to dng. Mt on DNA mang

mt gen bit c la chn v s dng nh mt mu d nhn dng (v d: bng cch lai khun lc) cc on khc, l cc on chng ln nhau cha cng mt gen. Sau , trnh t nucleotide ca cc on ny s c phn tch v nh vy c th xc nh c ton b cc on ca nhim sc th. T , bn ca mt vng c bit s c xy dng dn dn. Chu trnh sinh tan (lylic cycle). Mt kiu chu trnh sng ca thc khun th (bacteriophage) khi n xm nhim vi khun, iu khin cc hot ng sinh sn v sinh trng bng cc gen ca n v sinh ra cc bacteriophage th h con, chui ra khoi t bao vi khun sau khi pha v t bao o. Chu trinh tim tan (lysogenic cycle). L hin tng h gen ca bacteriophage hin din trng thi n nh v khng sinh tan trong t bo vt ch sng ca n. Cc t bo vt ch c th tip tc sinh trng v phn chia, v s sao chp ca h gen bacteriophage (prophage) c phi hp vi nhim sc th ca vt ch sao cho khi t bo phn chia th prophage cng c chuyn vo trong c hai t bo con. Prophage c duy tr bng cch hoc hp nht trong nhim sc th vt ch (v d: bacteriophage , bacteriophage 105) hoc nh l mt plasmid bn ngoi nhim sc th (v d: bacteriophage P1 v bacteriophage F116). T bo vt ch c th hoc khng th biu hin ra mt kiu hnh bin i. Chui contig (contiguous sequence). Mt oan trnh t dai c hinh thanh t mt s cac oan phn t ngn chng ln nhau (overlapping). Chui kham (concatemer). Phn t DNA bao gm nhiu oan ca bit ni vi nhau thng qua cac u dinh. Chui m ha (coding sequence). on phn t DNA mang m di truyn xc nh phin m thnh mRNA v sau dch m thnh chui polypeptide. Chuyn gen (transgenic). Qu trnh chuyn mt oan DNA ngoi lai (foreign DNA) bng cc k thut khc nhau (Agrobacterium, vi tim, bn gen, xung in...) vo mt c th vt ch (vi sinh vt, ng vt hoc thc vt). Chuyn nhim (transfection). K thut a DNA phage hoc DNA virus vo cc t bo vt ch. Cosmid. Vector lai (hybrid vector) c cu thanh t cac oan trinh t ca plasmid va cac v tr cos (u dinh) cua bacteriophage . Cng ngh DNA ti t hp (DNA recombinant technology). H thng cc phng php phng th nghim cho php ct on DNA t mt sinh vt ghp ni vo DNA ca mt sinh vt khc to ra phn t DNA ti t hp. Phn t ny c a vo cc sinh vt khc nhau to ra nhng ging chng vi sinh vt, thc vt v ng vt mi c nhng phm cht c bit, p ng nhu cu ngy cng cao ca sn xut v i sng con ngi. Cng ngh ny c ng dng rng ri trong y hc, dc hc, nng nghip v nhiu ngnh cng nghip khc.

Cng ngh sinh hc (biotechnology). Theo ngha rng l cc qu trnh cng nghip c s dng vi sinh vt hoc cc t bo ng vt v thc vt (cng ngh sinh hc truyn thng). Theo ngha ph bin hin nay l nhng qu trnh sn xut s dng cc ging sinh vt mi, c to ra bi cng ngh DNA ti t hp (cng ngh sinh hc hin ai). Trong cng ngh sinh hc truyn thng (ln men san xut ru, bia, u chua thc phm, lam phomt, trng trot, chn nui...) trc tin con ngi chon la cac i tng san xut thch hp (chung vi sinh vt, cy trng v vt nui) thng qua thc tin san xut va sau nay bng cac phng php khoa hoc nh gy t bin, phn lp Ngay nay, bng cach thay i gen nh cng ngh DNA tai t hp ngi ta a tao ra cac i tng san xut thch hp hn, c th thay i ca v lng va cht nhiu qua trinh san xut bng cng ngh sinh hc truyn thng trc y, nng chung ln v tr cao hn va m ra nhng trin vong ln cho cac linh vc hoat ng cua cng ngh sinh hc. Deoxyribonucleotide triphosphate (dNTP). Tin cht a c triphosphoryl ha (nng lng cao) cn thit cho qua trinh tng hp DNA. N c k hiu cho mt trong bn nitrogen base (A, G, T hoc C). Deoxyribonuclease (DNase). Loai enzyme nuclease thy phn (phn hy) DNA si i hoc DNA si n. Deoxyribonucleic acid (DNA). DNA la ai phn t sinh hoc co cu truc xon i, tn tai chu yu trong nhn t bao, trn cac nhim sc th, mang thng tin di truyn cua sinh vt. Phn t DNA gm hai chui polynucleotide, chui no xon quanh chui kia tao nn chui xon kep. Trong cac nucleotide, theo chiu doc cac gc phosphate ni xen ke vi cac phn t ng deoxyribose tao nn b khung bn ngoai cua chui xon kep, theo chiu ngang mi phn t ng u kt hp vi mt trong bn nitrogen base: adenine, guanine, cytosine hoc thymine. DNA khng trc tip th hin chc nng sinh hoc ma gian tip qua protein do no ma ha. DNA tao RNA, RNA tao protein. RNA cung la acid nhn (nucleic acid). No co thanh phn cu tao kha ging DNA, ngoai tr gc thymine (T) trong DNA c thay th bi gc uracil (U), va RNA dang si n ch khng phai dang xon kp nh DNA. Qua trinh oc ma di truyn cha trong DNA tng hp protein goi la s phin m (transcription) tao ra RNA mang thng tin di truyn l mRNA (messenger RNA). mRNA kt hp vi mt c quan t trong t bao la ribosome tao ra protein trong qua trinh dch ma (translation). Qua trinh trn c gi la qua trinh sinh hoc cn ban. Nm 1962, Watson (M) v Crick (Anh) chia s Gii Nobel vi Wilkins (Anh) v pht minh ra cu trc khng gian ca DNA v ngha ca n trong vic truyn thng tin di truyn. iu ng tic l Franklin, ngi c nhng ng gp ng k

cho pht minh ny mt trc . Theo qui nh th Gii Nobel khng dc php tng cho ngi mt. Dich chuyn im t (nick translation). Phng phap anh du DNA bng 32 phong xa [- P]dCTP nh enzyme DNA polymerase I ca E. coli. Dich ma (translation). S tng hp protein trn khun mRNA. Qu trnh chuyn thng tin di truyn trong trnh t base ca mRNA sang trnh t amino acid ca chui polypeptide trong t bo cn gi l qu trnh sinh tng hp protein. Dch m ngc (reverse translation). L k thut phn lp cc gen nh kh nng ca chng trong vic lai vi mt on m oligonucleotide no , on ny c chun b bng cch d on on m nucleic acid t nhng on m ha ca protein bit trc. Dideoxyribonucleotide triphosphate (ddNTP). Mt ng phn cua dNTP dung kt thuc mt chui DNA trong cac thi nghim xac inh trnh t gen (sequencing). Dimer. La mt hn hp c tao ra gia hai phn t ng nht nhng khi lng phn t thi gp i so vi phn t nguyn thy. DNA b sung (complementary DNA, cDNA). DNA c tng hp trn khun mu mRNA nh qu trnh phin ma ngc (reverse transcription). Do vy, no co trinh t sp xp cac nucleotide b sung vi trinh t cac nucleotide trn mRNA. V du: trn mRNA trinh t o la UUGAAG thi trn cac DNA b sung se co trinh t tng ng l AACTTC. DNA b sung c tng hp t nhin trong chu trinh sng cua virus mang vt cht truyn la RNA. V d: HIV, virus cum va cac retrovirus noi chung. DNA b sung c tng hp nhn to trong cac phong thi nghim xy dng th vin cDNA (cDNA library). DNA khun mu (template DNA). Si DNA ma trinh t cac nucleotide cua no c dung lam khun mu tng hp nn si DNA mi trong qua trinh ti bn (sao chp) hoc khuch i DNA (PCR) hoc tng hp nn si RNA mi trong qua trinh phin ma. DNA polymerase. Enzyme tng hp ban sao DNA trn khun mu DNA bng cch xuc tac phan ng gn tng nucleotide ring bit vao u chui DNA ang c tng hp. Nm 1959, hai nh khoa hc ngi M l Kornberg v Ochoa c nhn Gii Nobel v nhng nghin cu lm sng t c ch c bn ca qu trnh sao chp DNA lin quan n DNA polymerase I. DNA siu xon (supercoiled DNA). DNA xon li trn bn thn n, thng l kt qu ca s gp khc, m xon hoc xon li ca chui xon kp DNA. DNA v tinh (satellite DNA). L nhng on DNA mang cc trnh t lp li ni tip c thnh phn khc vi tr s trung bnh ca DNA h gen. DNA v tinh tao thanh bng theo gradient ty trong v d dang phn bit vi bng cua phn ln DNA h gen do

DNA v tinh co ty trong nho hn. Ban sao cua DNA v tinh lp lai hang triu ln trong h gen, tp trung chu yu vung tm ng va hai u cua nhim sc th. Dng (clone). Tp hp cc t bo hoc phn t ging ht nhau cng bt ngun t mt t bo hay phn t ban u. Dot blot. L ky thut trong o cac vt tron nho (hoc cac im) co cha nucleic acid c t ln mang nitrocellulose hoc nylon lai vi on mi DNA c nh du ng v phng x. a hinh dai cac oan ct han ch (restriction fragment length polymorphism, RFLP). Tnh a hnh chiu di cc on ct hn ch ch cc sai bit di truyn v tr nhn bit ca cc enzyme hn ch (chng hn nh do s thay i mt nucleotide) dn n s sai bit trong chiu di ca cc on hnh thnh t phn ng ct hn ch DNA vi cng mt enzyme. RFLP thng c dng thit lp bn di truyn vi mt s marker di truyn bit trc. anh du ui (end labelling). B sung phn t phong xa vao ui cua mt polynucleotide nh enzyme T4 polynucleotide kinase. u bng (blunt end). Cac u cua DNA si i khng co cac u 3 hoc 5 li ra (protruding ends). u dinh (cohesive ends hoc sticky ends). Cac u cua phn t DNA co cac trinh t b sung ngn co th dinh kt lai ni hai phn t DNA vi nhau. Cac u dinh thng do cac enzyme han ch tao ra. u tn cng C (C terminus). Gc carboxyl (COOH) t do v tr tn cng cua mt phn t protein hoc chui polypeptide. u tn cng N (N terminus). Gc amine (NH2) v tr tn cng ca mt phn t protein hoc chui polypeptide. Tt c cc polypeptide u c tng hp t u tn cng N n u tn cng C. im t (nick). im t gy mt si n trn DNA si i. in di trn gel (gel electrophoresis). K thut dng phn tch cc phn t nucleic acid hoc protein da vo s dch chuyn ca chng trn gi th dng gel (agarose hoc polyacrylamide) di nh hng ca in trng. S dch chuyn ca cc phn t ny ph thuc vo in tch, cu hnh, kch thc v khi lng phn t ca nucleic acid hoc protein cng nh dung mi v nng ca cht dng lm gi th. on ct hn ch (restriction fragment). Cc on DNA nh c sinh ra sau khi x l on DNA ln bng enzyme hn ch. on kt thc phin m (terminator hay transcription terminator). Trnh t nucleotide nm cui gen hot ng nh mt tn hiu kt thc s phin m. N ra hiu cho RNA polymerase gii phng phn t RNA mi c to thnh ra khi gen.

Lu khng c nhm vi cc b ba kt thc (terminator codons hay stop codons: UAG, UAA v UGA), xut hin trong mRNA, l tn hiu dng ca s dch m (xem m v ngha). C hai loi terminator ph bin: Rho-independent terminator (thng l mt cu trc thn-quai (stem-loop structure) trong RNA c phin m) nm u ca cc operons, v Rho-dependent terminator (vng khng c cu trc c trng ca RNA, khi khng c dch m, n c xem nh l yu t Rho) l nguyn nhn gy ra chiu phn cc ca s dch m (translational polarity). oan Klenow (Klenow fragment). Cn gi l oan ln cua DNA polymerase I. y l mt oan cua DNA polymerase I (khi lng phn t 76.000) ca E. coli bi mt hoat tinh exonuclease 53. on mi (primer). Mt trnh t DNA hay RNA ngn, bt cp vi mt mch ca DNA khun mu v c mang mt u 3-OH t do gip DNA polymerase bt u tng hp mt chui DNA mi. on nhi (stuffer fragment). Cn gi l vng m hay vng trung tm. L mt phn ca phage c th c loi b v thay th bng on chn DNA (insert DNA) m khng nh hng n kh nng sinh sn ca phage trong t bo vt ch. oan xui ngc nh nhau (palindrome). on DNA mch kp c trnh t sp xp cc base trn hai mch n ging ht nhau nu cng c c theo mt chiu (chng hn 53). V d: cc on nhn bit ca enzyme hn ch. ng du (replica plating). Phng php chuyn nguyn mu cc khun lc hoc vt tan t mt a thch gc sang a thch mi bng cch dng mng nylon (v d: mng Hybond-N+) va kht p ln mt thch ca a gc dnh ly cc t bo trong cc khun lc (colony) hoc vt tan (plaque) ca a gc, ri a mng ny p ln mt thch mi. n vi phin ma (transcriptional unit). oan DNA ma ha cho phn t RNA, bt u t im khi u phin ma n im kt thc phin ma, n c th di hn mt gen. n v sao chp (replicon). on DNA bt u t im khi u sao chp ko di v hai pha ti hai im kt thc sao chp. n v ti t hp (recon). on DNA ca gen c chiu di ngn s trao i cho khng th din ra bn trong n c na. Hin nay, c bit l mt cp nucleotide. ui polyA (polyA tail). on trnh t di 50-200 nucleotide adenine c b sung vo u 3 ca hu ht cc mRNA eukaryote sau khi phin m. E. coli (Escherichia coli). Vi khun thng c trong rut non ca ng vt c xng sng. E. coli c coi nh sinh vt mu cho vic nghin cu hot ng ca t bo. y l vi khun Gram m c kch thc genome khon 4106 base-pair. Cc qu trnh biu hin gen (phin m v dch m) i i vi nhau, sinh ra si mRNA c tng

hp mi v c s dng ngay cho qu trnh dch m. Khng c hin tng bin i sau dch m (post-translation). V th, E. coli c xem l mt trong nhng t bo vt ch n gin nht. Rt nhiu th nghim to dng gen ang c thc hin hng ngy ti cc phng th nghim u s dng E. coli lm vt ch vi nhiu chng khc nhau v mt di truyn v cho nhng ng dng c bit. Endonuclease. L enzyme nuclease ct bn trong phn t nucleic acid, ngc vi exonuclease la enzyme phn giai DNA t mt u hoc ca hai u. Nuclease thy phn nhng lin kt phosphodiester gia cc nucleotide ca mt phn t nucleic acid. Cc nuclease c th c hiu i vi DNA (deoxyribonuclease) hoc c hiu i vi RNA (ribonuclease). Enzyme. Cht xc tc sinh hc, l cc phn t sinh hoc co ban cht protein ong vai tro cht xuc tac cho cac phan ng bin i ha sinh. Enzyme gn DNA (DNA ligase). Enzyme dung ni cac phn t DNA vi nhau bng cach tao ra mi lin kt phosphodiester gia nhom 5-phosphate va nhom 3 hydroxyl trong qua trinh ti bn hoc sa cha DNA. Enzyme han ch (restriction enzyme, RE). Loai endonuclease co kha nng ct DNA tai nhng oan trinh t nht nh m chung nhn bit. Enzyme hn ch c pht hin vo nm 1970, chng tn ti trong t bo vi khun, c tc dng ct DNA ngoi lai (v d: DNA ca phage) ti nhng im xc nh, tiu dit DNA ny. Cho n nay hn 900 enzyme hn ch c tm thy. Cc enzyme hn ch c s dng rng ri trong cc phng th nghim thao tc gen nh nhng chic ko ct DNA ti nhng im c hiu. V tr ct ph thuc vo loi enzyme hn ch c la chn. Nm 1978, Arber (Thy S), Nathans (M) v Smith (M) c nhn Gii Nobel nh pht hin ra enzyme hn ch v nhng ng dng ca chng gii quyt nhiu vn quan trng ca sinh hc phn t. Cc enzyme ny l nhng chic ko phn t c th ct DNA thnh nhng on xc nh, m ra mt thi k pht trin mi ca sinh hc hin i-Thi k thao tc gen. Enzyme phin ma ngc (reverse transcriptase). DNA polymerase phu thuc RNA (RNA-dependent DNA polymerase) co trong cac RNA virus (retrovirus) c dung tng hp cDNA trong iu kin in vitro. Exon. Cc on DNA trong gen c chc nng phin m. Exon tn ti c sinh vt prokaryote v eukaryote. Ring sinh vt eukaryote cc exon nm xen k vi cc on intron. Cc intron chim ti 90% tng s DNA ca t bo eukaryote v khng c chc nng phin m. Eukaryote. Sinh vt c t bo mang nhn in hnh (nhn tht) ngha l nhn c bao bc bi mng nhn v tham gia vo hai c ch phn bo quan trng l nguyn phn v gim phn.

Exonuclease. Loi enzyme nuclease ch tc ng vo u tn cng ca phn t nucleic acid, ct ra tng nucleotide mt theo thi gian. Chng c th chuyn ha theo u 5 hoc 3 ca si DNA. cc Ex vivo. Thut ng dng ch cc th nghim thc hin trn t bo nui cy, t bo ny sau s c a vo mt c th sng.

-galactosidase. Enzyme c m ha bi gen lacZ. Enzyme ny thy phn lactose thnh glucose v galactose. Gen (gene). L n v di truyn, yu t quyt nh mt tnh trng c th. Thng tin di truyn ca cc gen c m ha trong DNA quyt nh tnh bin d ca loi v ca c th. DNA l mt chui bao gm cc n v nucleotide, c bn loi nucleotide mang bn nitrogen base khc nhau l adenine (A), guanine (G), cytosine (C), v thymine (T). Trnh t cc nucleotide ca mt gen xc nh mt polypeptide hoc mt RNA. Gen c kh nng b t bin. Cc gen ch yu nm dc theo nhim sc th trong nhn t bo. Mi gen chim mt v tr xc nh trn nhim sc th gi l locus. Gen c th tn ti nhiu dng gi l cc allele. Cc gen biu hin thng qua cc phn t do chng sinh ra l RNA (trong qu trnh phin m) v protein (trong qu trnh dch m). Gen ch th (reporter gene). L mt gen m ha m sn phm ca n c trc nghim mt cch d dng (v d chloramphenicol acetyltranferase). Gen ch th c th c gn vi bt k mt promoter no sao cho s biu hin ca n c th c dng th nghim chc nng ca promoter. Gen lacZ. Gen ca E. coli m ha -galactosidase thch hp cho chn lc th bin np bng khun lc xanh (-galactosidase s kt hp vi IPTG v X-gal c b sung trong mi trng nui cy) v khun lc trng (on DNA ngoi lai xen vo gia gen lacZ lm cho gen ny mt hot tnh v th khng sn xut c galactosidase). Ghp i lch (mismatch). S ghp i khng ng vi quy lut b sung gia cc nucleotide thuc hai si n DNA trong mch kp. Ghp exon hay splicing (RNA). Qu trnh ct b nhng intron v ni cc exon ca sn phm phin m ban u (tin thn mRNA) to thnh mRNA hon chnh (mature mRNA). Qu trnh bin i ny xy ra trong nhn t bo. Gc ti bn (origin, ori). Trnh t nucleotide hoc v tr trn DNA m bt u s ti bn (sao chp). Gradient. Bin thin ca mt i lng theo mt hng no . Mt gradient mt c xc lp trong mt s trng hp ly tm. Mt gradient proton hoc ion c to ra qua mt mng nh s vn chuyn tch cc i hi nng lng.

H gen (genome). L tp hp cc gen c trong mt t bo n bi eukaryote, trong mt t bo prokaryote hoc trong mt virus. H gen cha toan b DNA cua c th, vi du: h gen ngi cha DNA dai khoang 1,6 m nhng chi rng khoang 5 phn ti mm. ngi, s DNA noi trn c chia lam 46 phn co dai ngn khac nhau goi la nhim sc th (chromosome), la tp hp DNA dang nen cht n kich thc ng kinh chi con 3-4 phn triu met. Tuy nho nh vy, nhng nhim sc th ngi co n 3 t gc nucleotide. S sp xp c thu cua chung quyt inh ban cht sinh hoc cua c th. Hot tnh phng x c hiu (specific radioactivity). L hot phng x trn mt n v nguyn liu, chng hn: mt mu d nh du phng x c th c hot tnh c hiu 106 ln m/pht trn microgram. Hot tnh c hiu cng c dng xc nh hot ca enzyme. Hunh quang (fluorescence). Hin tng pht mt sng nh sng c bc sng khc vi bc sng c hp th trc . Mt s phn t c gi l th hunh quang (v d: enzyme luciferase con om m) do c c tnh ny. In du DNA (DNA fingerprinting) hay in du di truyn (genetic fingerprinting). L phng php dng cc mu d phng x hoc dng k thut PCR nhn dng cc bng DNA c cc on lp li vi tn s cao. Bn mu hnh cc bng DNA l duy nht i vi mi c th, v do vy c th dng xc nh c trng c th hoc quan h huyt thng. In du chn DNA (DNA footprinting). Phng php nhn dng cc vng DNA m cc protein iu ha bm vo. Intron. Nhng on DNA nh sinh vt eukaryote khng mang thng tin m ha amino acid, phn b ri rc dc theo phn t DNA. Sau khi thng tin t DNA c phin m sang mRNA th cc intron trn mRNA b ct b, cc on mRNA cn li gm ton cc exon c ni li vi nhau v chuyn n ribosome dng lm khun mu cho qu trnh dch m. Intron khng thy c sinh vt prokaryote. In vitro v in vivo. La thut ng m ta thi nghim trong ng nghim (in vitro) va trong c th (in vivo). Cung vi s pht trin ng dung cua may tinh, hin nay cac nha khoa hoc con tin hanh thi nghim m phong bng computer. Qua trinh nay goi la thi nghim in silico. Ko di on mi (primer extension). S tng hp mt bn sao nucleic acid bt u t on mi. c s dng nh du phng x on DNA lm mu d hoc khuch i mt on DNA bng k thut PCR. Khng nguyn (antigen). Phn t thng tm thy trn b mt t bo, c tc dng kch thch s to thnh khng th. Do vy, n c dng gy nn mt phn ng min dch.

Khng th (antiboby). Mt protein (immunoglobulin) do bch cu lympho B ca h thng min dch sn sinh, c tc dng nhn bit mt khng nguyn ngoi nhp c hiu v gn vi n, nu khng nguyn nm trn b mt t bo th vic gn kt ny s dn ti s kt cm t bo v lm bt hot khng nguyn. Khng th n dng (monoclonal antibody). Khang th xut hin trong phan ng min dich co nhim vu gn va tham gia loai bo cht la (antigen) lot vao c th. Thng thng, trong phan ng min dich co mt hn hp ca nhiu loi khang th. Tuy nhin, nh t bao lai hybridoma ngi ta co th tao ra mt loai khang th goi la khang th n dong. Khang th n dong chu yu c s dung cho muc ich chn oan bnh. Khuch i (amplification.) S sn xut nhiu bn sao ca mt trnh t DNA nh k thut PCR. Khung c m (open reading frame, ORF). L mt trnh t m ha chui polypeptide c bt u vi m khi u (initiation codon) v kt thc bng mt m dng (stop codon). Mt khung c m b ngn chn nu mt stop codon c nh v gn vi m khi u. Mc d v l thuyt ma di truyn c xy dng da trn b ba nucleotide, do s co ba khung c co th co trn mi si DNA, tuy nhin trong thc t khung c chnh xc c xc nh bi mt im bt u c nh. Khuyt on (deletion, deficiency). t bin nhim sc th dn n lm mt mt on vt cht di truyn v thng tin di truyn cha trong n ri khi nhim sc th. Kiu hoang di (wild type). Dng thng thy nht ca mt gen trong qun th hoang di. Allele kiu hoang di c k hiu bng mt ch in hoa hoc thm du cng sau ch vit thng, v d: A hay a+. Allele kiu hoang di thng l tri v cho kiu hnh bnh thng. Kilobase (kb). 1000 base (hoc cp base), c dng nh n v o hoc xc nh chiu di ca cc phn t DNA hoc RNA. Kinase. Cc enzyme xc tc phn ng phosphoryl ha mt phn t nhn nh ATP. K thut di truyn (genetic engineering). Cn gi l cng ngh DNA ti t hp. Bao gm h thng cc phng php di truyn phn t dng thao tc vt cht di truyn, vi ba bc chnh gm ba khu chnh. 1) Tch chit DNA t nhng sinh vt khc nhau; 2) Ct v ni DNA nhng im c hiu to ra DNA ti t hp (DNA mang cc gen c ngun gc khc nhau), v d: DNA plasmid c mang gen ca ngi; 3) a DNA ti t hp vo hot ng trong cc t bo hoc c th sng sinh ra nhng sn phm c bit cn thit cho con ngi, v d: DNA plasmid mang gen to insulin ca ngi c a vo vi khun E. coli sn xut.

Lai khun lc (colony hybridization). Ky thut lai in situ xac inh vi khun mang vector khm (chimeric vector) ma DNA cua vector nay tng ng vi mt oan ma ha c bit nao o. Lai phn t (molecular hybridization). Qu trnh trong hai mch nucleic acid b sung (A-T, G-C) bt cp hnh thnh nn mt mch kp. y l mt k thut hu ch pht hin mt trnh t nucleotide chuyn bit. Lai ti ch (in situ hybridization). Qu trnh bt cp gia mu d (l mt trnh t DNA si n hay RNA) vi DNA ca t bo c c nh trn lam knh. Lp bn hn ch (restriction mapping). K thut dng xc nh v tr cc im ct hn ch trn phn t DNA. Linker. Mt oligonucleotide tng hp c hai u bng, cha mt hoc nhiu vi tri ct han ch cho php ni cDNA si i vi cac plasmid vector hoc bacteriophage vector. cDNA si i trc c x ly vi DNA polymerase cua bacteriophage T4 hoc DNA polymerase I cua E. coli to u bng. Cc linker sau khi gn vi hai u bng ca on cDNA nh DNA ligase s c ct hn ch to ra u so le tng ng vi hai u ca vector. Phn ng gn gia on cDNA c mang linker hai u vi vector cng c xc tc nh DNA ligase. Lysosome. Mt bo quan c mng bao bc trong t bo cht ca nhng t bo eukaryote. Lysosome cha nhiu enzyme thy phn. Ly tm theo gradient mt (density gradient centrifugation). K thut tch cc hp cht da vo s khc nhau v mt ca chng c thc hin bng phng php ly tm lm lng cc cht qua mt gradient nng ca saccharose hoc CsCl. M di truyn (codon). Nhm ba nucleotide nm k nhau (b ba) trn phn t mRNA xc nh mt amino acid trn chui polypeptide, hoc l tn hiu kt thc vic tng hp polypeptide. M thoi bin (degenerate codon). M di truyn m mt amino acid c quy nh bi mt s b ba nitrogen base, ch khng phi ch bi mt b ba. Thoi bin l c im vn c ca m di truyn tn ti ph bin sinh gii. Ma v nghia (nonsense codon) hay m dng (stop codon). L codon ma o qua trinh dich ma dng lai (ni kt thuc cua chui polypeptide). Co tt ca ba codon v nghia vi cac tn goi la amber (UAG), ocher (UAA) va opal (UGA) Maturation. Qu trnh trong cc mRNA va c phin m tri qua mt s bin i ha hc tr thnh mRNA hon chnh sn sng lm khun mu cho vic tng hp protein. My m nhp nhy (scintillation counter). My dng xc nh hot tnh phng x trong mt mu th nghim.

Mu d (probe). Mt on RNA hay DNA chuyn bit c nh du bng ng v phng x hay bng ha cht (cht pht hunh quang hoc enzyme), dng nh v mt trnh t nucleic acid nht nh thng qua k thut lai phn t (xem Northern blot, Southern blot...) Mt quang (optical density). Thng s cho php o hp th nh sng mt bc sng no ca mt mi trng hoc dung dch. Monomer. L cc phn t n v nh, c th lin kt vi cc phn t n v ging n hnh thnh nhng phn t ln hn (polymer). V d: cc nucleotide l cc monomer ca nucleic acid v cc amino acid l monomer ca protein. Nm men Saccharomyces cerevisiae. L mt vi sinh vt nhn tht c s dng nhiu trong cng ngh DNA ti t hp. Genome ca nm men S. cerevisiae khong 1,35107 base-pair nhiu hn E. coli khong 3,5 ln. Nm men thng c dng lm t bo vt ch biu hin nhng protein c cu trc phc tp cn qu trnh hu dch m m vi khun E. coli khng th p ng. Nhn t kim sot phin m (transcription control element). on nucleotide nm xung quanh im bt u v kt thc ca mi gen, tham gia vo s iu ha hot ng biu hin ca gen. Nhit nng chy (melting temperature, Tm). L nhit m c mt na s phn t ca mt trnh t DNA b bin tnh. Northern blot. K thut chuyn v c nh RNA t formaldehyde agarose gel (sau khi c phn on bng in di) ln mng lai bng nylon hoc nitrocellulose lai vi mu d c nh du ng v phng x [-32P]dCTP hoc digoxigenin-dUTP. Nucleic acids. Nhng polynucleotide sinh hc thin nhin, trong nhng n v nucleotide c kt hp vi nhau bi nhng lin kt phosphodieste thnh trnh t DNA hoc RNA ring bit. Nucleoside. Mt hp cht gm mt base purine hoc pyrimidine kt hp ng ha tr vi mt phn t ng pentose. Nucleotide. Mt nucleoside phosphoryl ha vi mt trong nhng hydroxyl ca pentose. Phn t ng vai tr cu trc c s ca DNA v RNA, gm ba phn: ng pentose (ribose trong RNA, deoxyribose trong DNA), nitrogen base v gc phosphate. Nucleolytic. Phan ng thy phn mt cu ni phosphodiester trong mt nucleic acid. Nuclease Bal 31. Mt loi enzyme exonuclease thy phn c hai si ca phn t DNA cng mt lc. Oligo. Tip u ng c ngha l t, v d: oligonucleotide (polynucleotide) c t nucleotide hoc oligopeptide (polypeptide) c t peptide.

Oligo(dT)-cellulose. Mt on ngn gm cc gc deoxy-thymidine lin kt vi c cht cellulose, c s dng tinh sch mRNA eukaryote bng phng php sc k ct. Oligomer. Thut ng chung ch mt on ngn cc monomer. Oligonucleotide. Mt on ngn cc monomer l nucleotide, thng t 20-30 nucleotide. n ha (temperate). Trng thi tim tan ca cc bacteriophage t bo vt ch. -peptide. Mt phn ca protein -galactosidase, c m ha bi on gen lacZ. Phage. Vit tt ca bacteriophage (thc khun th), l loi virus xm nhim v sinh sn bn trong vi khun. Phage thng c v bc protein, phc hp bao gm phn u c hnh a din cha nucleic acid v ui m qua nucleic acid xm nhp vo vi khun ch. Sau qu trnh nhn ln ca nucleic acid ca phage, t bo vi khun ch thng b tan bin. Loi phage lun lun lm tan t bo vi khun khi chng xm nhim vi khun gi l phage c. V d: phage T4. Ngc li, cn c phage n ha, khi xm nhim vi khun n gy nn phn ng tim tan, ngha l h gen ca phage gn vo nhim sc th vi khun v c sao chp cng vi nhim sc th . H gen ca phage trng thi gn nh vy vi nhim sc th vi khun gi l prophage. Phagemid. L mt loi plasmid vector c mang cc on trnh t ca phage. Phn ng chui polymerase (polymerase chain reaction, PCR). Phng php dng trong phng th nghim khuch i cc on DNA c bit ln hng triu ln trong vng vi gi thng qua 20-30 chu k nhit, mi chu k bao gm ba mc nhit : bin tnh 90-95oC, bt cp vi mi 40-65oC hoc hn v tng hp mch mi nh DNA polymerase chu nhit (Taq polymerase) 70-72oC. PCR c ng dng rng ri trong chn on y hc, phn tch s a dng sinh hc, chn ging v trong nhiu lnh vc khc. Nh khoa hc M (Tin s Mullis) ngi pht minh ra k thut PCR nhn gii Nobel nm 1993. Cng chia s Gii Nobel vi Mullis l Smith (Canada) do c nhng ng gp mang tnh nn tng cho vic gy t bin im nh hng, da trn cc oligonucleotide v vic pht trin chng trong cc nghin cu protein. Phn tch trnh t gen (gene sequencing). L k thut xc nh trnh t theo cu trc bc mt ca chui cc nucleotide trong mt phn t nucleic acid. Phn tch trnh t ca DNA c cc phng php ha hc ca Maxam-Gilbert v phng php enzyme ca Sanger. Trong nhng nm gn y, mt s phng php xc nh trnh t mi nh s h tr ca my tnh xut hin. Bn cnh k thut thng thng s dng cc polyacrylamide gel phn ly cc phn t DNA c di khc nhau, cc k thut mi lin quan n pht hin hunh quang ca cc nucleotide c nh du, phn tch trnh

t DNA bng khi ph, in di mao dn hoc lai vi cc on oligonucleotide c tng hp nhn to cng ra i. Nm 1980, Sanger (Anh) v Gilbert (M) c trao gii Nobel do c nhng ng gp quan trng v phng php xc nh trnh t cc nucleotide trong phn t DNA. ng gp ny l mc lch s to ln trong sinh hc phn t, l nguyn l ca tt c cc my xc nh trnh t DNA t ng ang s dng hin nay trn khp th gii. Phn cui (telomere). on cui, phn cui ca mt nhim sc th thng ca eukaryote, bao gm nhng trnh t DNA ngn c lp li nhiu ln. Phn tm (centromere). Phn co tht c thy trn nhim sc th k gia, l ni hai nhim sc t nh vi nhau. Phin m (transcription). L qu trnh c xc tc bi enzyme phin m RNA polymerase tng hp mRNA t khun mu DNA. Phin m ngc (reverse transcription). Qu trnh tng hp DNA t khun mu mRNA nh enzyme phin m ngc (reverse transcriptase). Phng x t ghi (autoradiography). K thut pht hin cc phn t c nh du phng x thng qua hiu ng to nh ca cc phn t ny trn phim X-quang. Phosphatase kim (alkaline phosphatase). Enzyme loi b cc nhm 5phosphate t u ca cc phn t DNA v li cc nhm 5-hydroxyl. Plasmid. La DNA vi khun c cu trc mch vng kp, nm trong t bo cht v ngoi nhn, c kh nng sao chp c lp i vi nhim sc th ca t bo. Tn ti c sinh vt prokaryote v eukaryote. Ngy nay, cc plasmid thit k nhn to c s dng rng ri nh l cc vector dng trong cc k thut to dng v biu hin gen. Plasmid khng tip hp (non-conjugative plasmid). V d: plasmid ColE1, l loi plasmid khng dng s tip hp cho qu trnh sng, thng thng l cc plasmid c kch thc b, tn ti vi mt s lng nhiu. C ch nhn ln (nhiu bn sao) ca chng cng khc hon ton vi plasmid tip hp. Plasmid khng tng hp (incompatible plasmid). L nhng plasmid c th cng tn ti vi nhau trong mt vi th h t bo vt ch, sau trong qu trnh phn chia ca t bo, mt s trong chng s b thi loi. Mun tng hp trong cng mt t bo vt ch, cc plasmid khc nhau phi c chung mt s c im trong qu trnh tn ti. Plasmid tip hp (conjugative plasmid) hay plasmid F (fertility (F) plasmid). L cc plasmid chuyn giao nhng bn sao DNA ca chng t vi khun ny sang vi khun khc nh phng thc tip hp (do chng c t hp gen sn xut cc ng protein hay cn gi l lng gii tnh (sex pile) lm cu ni gia hai t bo vi khun vi nhau). DNA ca plasmid, thm ch c DNA ca h gen vi khun, thng qua cc ng protein ny chuyn t t bo vi khun cho sang t bo vi khun nhn. Cc plasmid, ngoi vic chuyn giao DNA ca ring chng, cn c kh nng chuyn giao

mt phn hay nhiu phn h gen ca t bo vi khun vt ch n t bo vi khun nhn khc. Do vy, chng c gi l cc nhn t gii tnh (sex factor) ca vi khun vt ch, c vai tr quan trng trong bo tn di truyn ca vi khun theo phng thc ny. Plasmid tng hp (compatible plasmid). L nhng loi plasmid c trong cng mt t bo vt ch nhng khng nh hng ln nhau. Khi t bo vt ch phn chia, chng cng ng thi phn chia v tn ti vnh vin. Polyacrylamide. L polymer ca acrylamide v bisacrylamide c cu trc gm cc lin kt cho to ra mt mng xp (ging bt bin). Cc cht phi chui vo l gel mi ra c, v vy nhng cht no c khi lng phn t nh s ra trc v ngc li. Polynucleotide. Trnh t nhng nucleotide ni ng ha tr vi nhau, trong v tr 3 ca pentose ca mt trong nhng nucleotide c ni vi mt lin kt phosphodieste v tr 5 ca pentose ca nucleotide tip theo. Polypeptide. Mt chui di nhng amino acid ni vi nhau bi nhng lin kt peptide. Prokaryote. Sinh vt n bo khng c nhn t bo in hnh, DNA nm trong t bo cht khng c mng bao bc, khng c nguyn phn v gim phn; i din in hnh l vi khun. Prophage. Phage n ha xen vo nhim sc th ca vi khun tim tan. N sao chp ng thi vi nhim sc th ca t bo vi khun ch. Protein. Mt phn t ln gm mt hoc nhiu chui polypeptide, mi chui c mt trnh t amino acid v mt khi lng phn t c trng. Protein l hp cht quan trng bc nht i vi c th sng. V cu trc, protein l phn t mch di gm cc n v cu trc nh l cc amino acid ni vi nhau qua mi lin kt peptide. Khi lng phn t ca protein t vi nghn n vi triu. C khong 20 loi amino acid. Cc loi protein phc tp hn c lin kt thm vi cc nhm b sung. Protein dung hp (fusion protein). L mt protein ti t hp lai c m ha bi mt gen lai (fusion gene) do s dung hp in vitro cc on gen khc nhau trn plasmid vector v sau bin np vo vi sinh vt ch (chng hn E. coli). V vy, protein dung hp s mang trnh t amino acid ca hai protein khc bit c tng hp t u N ca vector biu hin. Protein nguyn th (native protein). L mt protein ti t hp c m ha bi mt gen ngoi lai (foreign gene) trong vi sinh vt ch. Khc vi protein dung hp, protein nguyn th c tng hp t u N ca n ch khng phi t u N ca vector.

Purine. Mt hp cht d vng, kim, c nitrogen, l thnh phn ca nhng nucleotide v nucleic acid. Purine cha mt nhn pyrimidine kt hp vi mt nhn imidazol. Pyrimidine. Mt nitrogen base d vng c trong cc nucleotide v nucleic acid. Retrovirus. L loi virus RNA cha enzyme reverse transcriptase v sinh sn di dng DNA mch kp. Chng c kh nng xm nhim t bo vt ch cao. Khi xm nhim n c kh nng gn h gen ca virus vi h gen ca t bo vt ch, l c s thit k cc vector liu php gen hiu qu. Ribonuclease. Enzyme xc tc c hiu vic phn hy RNA bng cch ct cc mi lin kt phosphodiester trn RNA. Ribonucleic acid (RNA). Thng l phn t a phn mch n gm cc n v cu trc c s l ribonucleotide. V mt ha hc RNA rt ging vi DNA. RNA l vt cht di truyn cua mt s virus va la cac phn t trung gian trong qua trinh tng hp protein ma thng tin v trinh t amino acid cua chung a c ma ha trong DNA. Ribonucleotide. n v cu trc c s ca RNA, gm ba thnh phn: ng ribose, nitrogen base v nhm phosphate. Ribosome. La c quan t khi kt hp vi mRNA tao ra b may tng hp protein. Trong t bao thng co hang nghin ribosome, ribosome cua moi t bao u gm mt tiu n vi nho va mt tiu n vi ln. Mi tiu n vi co mang nhiu protein va rRNA (trong rRNA l thnh phn ch yu chim khong 65%) co kich thc khac nhau. Ngi ta cng thy ribosome trong ty th, c s tng hp mt s protein ty th. RNA b sung (complementary RNA). RNA sinh ra bng cach phin ma t khun mu si n DNA tng ng. RNA ligase. Enzyme ni cac oan RNA vi nhau sau khi cac intron c ct ri khoi tin cht cua mRNA (pre-mRNA) cac sinh vt eukaryote, tao ra mRNA hon chnh sn sang tham gia vao qua trinh dich ma din ra trn ribosome. RNA polymerase. Con goi la RNA polymerase phu thuc DNA (DNA-dependent RNA polymerase), xuc tac vic tng hp RNA trn khun mu DNA trong qua trinh phin ma. RNA ribosome (ribosomal RNA, rRNA). La thanh phn c ban cua ribosome, ong vai tro xuc tac va cu truc trong tng hp protein. Ty theo h s lng rRNA c chia thanh nhiu loai: eukaryote co rRNA 28S; 18S; 5,8S va 5S; con cac rRNA E. coli co ba loai: 23S, 16S va 5S. rRNA chim nhiu nht trong bn loai RNA (khong 80% tng s RNA t bao), tip n la tRNA khong 16%, mRNA chi khong 2%. Ngoi ra, t bao sinh vt eukaryote con cha nhng phn t RNA kch thc nho ca nhn (small nuclear, snRNA) chim khong <1% tham gia vao ghep ni cac exon.

RNA thng tin (messenger RNA, mRNA). Mt loi RNA c phin m t mt trnh t DNA. mRNA truyn thng tin di truyn t nhim sc th ti ribosome tng hp protein. Trong qua trinh o mt si cua chui xon kep DNA c dung lam khun mu, doc theo no cc nucleotide cua mRNA b sung c xp thanh hang, ni vi nhau tao nn mt polynucleotide ging ht si DNA khng lam khun mu ngoi tr thymine c thay bng uracil. Qua trinh nay goi la phin ma v phn t mRNA mang ma di truyn c dung iu khin s hinh thanh protein trn ribosome. RNA vn chuyn (transfer RNA, tRNA) . Loai RNA mang cac amino acid n ribosome va sp xp chung doc theo phn t mRNA a nm sn o. Tai y, cac amino acid ni vi nhau bng lin kt peptide tao thanh phn t protein. Mi amino acid co mt phn t RNA vn chuyn ring vi b ba c trng v nh vy cac amino acid c sp xp theo trt t ca cac nitrogen base trn mRNA trong qua trinh dich ma. RNA kch thc nh ca nhn (small nuclear RNA, snRNA). Ngoi mRNA, tRNA V rRNA, t bao eukaryote con cha nhng phn t RNA kch thc nho ca nhn (chim khong <1%) tham gia vao ghep ni cac exon. Sng lc (screening). K thut nhn dng mt dng DNA trong mt th vin h gen (genomic library) hoc th vin cDNA (cDNA library) bng mt phng php lai mu d c nh du [-32P]dCTP vi cc vt tan (trng hp dng bacteriophage lm vector to dng v cho xm nhim vo vi khun E. coli) hoc khun lc (dng plasmid lm vector to dng) ca cc th vin trn mng nylon hoc nitrocellulose. Tn hiu lai c pht hin bng phng x t ghi trn phim X-quang. Sinh hc phn t (molecular biology). Khoa hc nghin cu cc hin tng sng mc phn t. Lnh vc khoa hc tr tui ny l im gp nhau ca cc khoa hc kinh in nh di truyn hc, ha sinh hc, t bo hc, vt l hc, ha hc hu c v ha l. Theo cch hiu ph bin hin nay, sinh hc phn t l khoa hc nghin cu cc gen v hot ng ca chng mc phn t, bao gm phin m, dch m, sao chp, iu ha biu hin gen, ti t hp v chuyn gen... Sinh tng hp protein (protein synthesis). Phn ng ha hc din ra trn ribosome to nn cc phn t protein t cc amino acid trn c s thng tin di truyn nhn c t trong nhn t bo thng qua mRNA. Southern blot. K thut chuyn v c nh DNA bin tnh t agarose gel (sau khi c phn on bng in di) ln mng lai bng nylon hay nitrocellulose lai vi mu d c nh du ng v phng x [-32P]dCTP hoc digoxigenin-dUTP. S bn sao (copy number). (1) S cc phn t plasmid c trong mt t bo vi khun. (2) S lng cc bn sao ca mt gen trong h gen ca mt sinh vt. do S phng x t ghi (autoradiogram). Hnh nh sinh ra trn phim X-quang s pht x ca cc h t phng x .

Ti t hp (recombination). Qu trnh m trong nhim sc th hay phn t DNA t ra ri cc phn t c ni li theo mt t hp mi. Qu trnh ny c th xy ra trong t bo sng (qua s trao i cho trong phn bo gim nhim) hay trong ng nghim nh cc enzyme ct v ni DNA. To dng gen (gene cloning). Cn gi l nhn dng, tch dng hay dng ha, l s sn sinh nhiu bn sao ca mt phn t DNA, thng l phn t DNA ti t hp trong plasmid vector, bng cch sao chp phn t trong mt vt ch thch hp chng hn vi khun E. coli. Terminal transferase. Enzyme b sung cc gc nucleotide vo u 3 ca oligonucleotide hoc polynucleotide. T bo kh bin (competent cell). Cc t bo vi khun c kh nng tip nhn DNA ngoi lai trong qu trnh bin np. Th bin np (transformant). T bo hoc sinh vt nhn c gen ca mt sinh vt khc trong qu trnh bin np v biu hin chc nng ca gen ra kiu hnh. Th a hinh (polymorphism). M ta s co mt ng thi cua qun th trong h gen biu hin bin di co tinh cht allele, co th quan sat trn cc allele tao ra cac kiu hinh khac nhau, hoc s thay i cua DNA anh hng n kiu ct hn ch (restriction patterns). Th ti t hp (recombinant). Cc c th hoc t bo mang cc t hp gen khc vi cha m ca chng do cc qu trnh ti t hp di truyn sinh ra. Thng tin di truyn (genetic information). Thng tin c lu tr trong cc phn t DNA ca sinh vt dng trnh t sp xp ca bn nucleotide k hiu l A, T, C v G ng vai tr nh nhng ch ci ca ngn ng di truyn. Trong ngn ng ny, mi t ch c ba ch ci gi l mt b ba. Ngha ca mi t l mt amino acid c mt trn phn t protein tng ng. Mi cu ca ngn ng di truyn l mt gen cha ng thng tin di truyn m nhim mt chc nng trn vn. Mi chc nng l mt c tnh sinh l, hnh thi hay cu trc ca s sng. Do c ch sao chp theo kiu na bo ton ca DNA m thng tin di truyn c truyn chnh xc t th h n sang th h kia hu nh khng thay i. Th vin cDNA (cDNA library). Tp hp cc dng DNA c to ra t mRNA ca mt t bo hoc mt m c th trong bacteriophage vector, i din cho thng tin di truyn m cc t bo biu hin. Th vin h gen (genomic library). Tp hp tt c cc on DNA c to ra t phn ng ct hn ch genome trong bacteriophage vector, i din c cho ton b cho thng tin di truyn ca mt h gen. Tr hon gel (gel retardation). Phng php xc nh im bm ca protein trn cc on DNA, da vo di chuyn chm ca chng so vi DNA khng b protein bm trong cc th nghim in di trn gel.

Trnh t dn u (leader sequence). Mt trong ba phn ch yu ca mt phn t mRNA. Trnh t ny nm u 5 ca mRNA v mang thng tin ribosome v cc protein c hiu nhn bit bt u qu trnh tng hp polypeptide, trnh t dn u khng c dch m thnh trnh t cc amino acid. Trnh t iu ha (regulatory sequence). Mt trnh t ca DNA tham gia vo qu trnh iu ha ca gen. V d: trnh t promoter hoc operator. Trnh t khi ng (promoter). Trnh t nucleotide c hiu nm trong thnh phn operon, c chc nng iu ha hot ng ca operon, ni RNA polymerase bm vo bt u qu trnh phin m. Trnh t c trng ca promoter c khong 20-200 nitrogen base. Trnh t Shine-Dalgarno (Shine Dalgarno sequence, SD). Cn gi l vng lin kt ribosome (RBS), l mt phn ca trnh t nucleotide u 5 ca mt mRNA prokaryote c th kt hp b sung cp base vi u 3 ca 16S rRNA, dng lm tn hiu cho s khi u dch m. Trnh t tng cng (enhancer). Trnh t nucleotide dng cis lm tng cng phin m ca promoter trong gen eukaryote. N c th nm cch promoter hng ngn cp base v hot ng theo c hai hng bt k v tr no so vi promoter. gn mi (annealing). Dung chi s bt cp cua khun mu DNA si n vi primer (mi) tng hp nn mt si DNA b sung bng cch s dng cc dNTP c trong mi trng ko di primer nh s xc tc ca enzyme Taq DNA polymerase (trong khuch i PCR) hoc DNA polymerase I (trong tng hp cDNA). c ch amber (amber suppresser). La cac gen t bin (ma ha cho tRNA) ma nhng anticodons cua no a c kich hoat co th nhn UAG codon cung nh cac codon trc o. Vt ch (host). T bo dng nhn cc phn t DNA ln nhiu ln. Vector. L cc phn t DNA c s dung trong tao dong v biu hin gen, va nhn ban chung trong t bo vt ch (E. coli hoc nm men). C ba nhm vector chnh gm: (1) Nhm plasmid, (2) Nhm phage/phagemid, v (3) Nhm nhim sc th nhn to (artificial chromosome: BAC, YAC). Y tng v vector chuyn gen bt ngun t cac plasmid cua vi khun. Vector chuyn gen la cc phn t DNA co kha nng t tai sinh, tn tai c lp trong t bao va mang c cac gen cn chuyn. Vector to dng (cloning vector). Phn t DNA mch kp c kh nng t sao chp trong t bo vt ch. C th gn vo phn t ny mt on hoc mt vi on DNA khc ngun to nn phn t DNA ti t hp dng nhn dng. Vector biu hin (expression vector). Phn t DNA mch kp c mang cc tn hiu cn thit (promoter, terminator v vng lin kt ribosome) cho s biu hin ca

mt khung c m (gen) s c nhn dng v sn xut protein tng ng trong t bo vt ch. Vt tan (plaque). Vng trn trong sut xut hin trn thm c ca cc vi khun mc trn mi trng thch c, do s tan v lp li nhiu chu k ca cc t bo vi khun b bacteriophage xm nhim v sinh tan. Virus. Phc hp cha nucleic acid (DNA hoc RNA) nm trong mt v bc protein, c kh nng gy nhim v ti bn bn trong t bo vt ch c hiu, to ra nhiu virus, lan truyn t t bo ny sang t bo khc. Virus l dng sng khng c cu trc t bo, c kh nng xm nhp vo cc t bo sng xc nh v ch sinh sn bn trong cc t bo . Ging nh tt c c sinh vt khc, virus c b my di truyn ca ring mnh, m ha vic tng hp cc ht virus t cc cht c trong t bo vt ch. Nh vy, virus l nhng vt k sinh ni bo. Virus phn b khp ni trong t nhin, xm nhp vo tt c cc nhm sinh vt. Ngi ta bit khong 500 loi virus xm nhp ng vt mu nng, 300 loi xm nhp thc vt bc cao. Mt s khi u ung th ng vt v ngi c th do virus. Virus tn ti hai dng: dng ngh hay ngoi bo v dng sinh sn hay ni bo. Kch thc ca cc ht virus t 15-350 nm, chiu di ca mt s loi virus c th t ti 2000 nm. Phn ln virus ch nhn thy c qua knh hin vi in t. Cht mang thng tin di truyn ca virus l nucleic acid: DNA hoc RNA. V vy, c th phn virus thnh hai loi: loi mang DNA v loi mang RNA. V tr cos (cos site) hay u cos (cos end). Trnh t nucleotide c ct ra to thnh cc phn m rng si n, kt dnh hai u cui ca cc phn t DNA mch thng ca mt phage no (v d: phage ). Vng cp tc (hairpin loop). Vng chui n b sung to np gp cha cc cp base to thnh xon kp, c dung lam mi (primer) cho qua trinh tng hp si cDNA th hai. Vng cng hng (downtream). cp n v tr ca mt on trnh t no nm pha u 3 ca gen hoc mt on gen quan tm. Vng lin kt ribosome (ribosome binding sites, RBS). L trnh t nuleotide trn phn t mRNA giup cho no bam vao ribosome trong qua trinh dich ma (xem trnh t Shine-Dalgarno). Vng ngc hng (upstream region). V tr ca mt trnh t nucleotide no nm pha u 5 ca phn t DNA so vi gen quan tm. Vng to dng (multiple cloning sites, MCS) hay vng a ni (polylinker hay polycloning sites). oan DNA ngn trong mt vector th h th ba (v d: cc nhm pUC, pGEM, pBluescript) co cha cac vi tri nhn bit cho mt s enzyme ct hn ch thng dng, c thit k chn on DNA ngoi lai vo y.

Western blot. K thut chuyn protein tng s c phn tch bng in di SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) ln mng nylon hoc nitrocellulose lai vi khng th mt c hiu v sau l khng th hai c nh du enzyme nhm pht hin protein khng nguyn tng ng ca n. YAC (Yeast artificial chromosome). Nhim sc th nhn to ca nm men, c dng lm vector to dng nhng on DNA c kch thc rt ln trong nm men.

Ti liu tham kho/c thm

1. Ban T in-NXB Khoa hc v K thut. 2002. T in Bch khoa Sinh hc. NXB Khoa hc v K thut, H Ni. 2. Lawrence E. 1995. Hendersons Dictionary of Biological Terms. 7th ed. Longman Group Ltd. Singapore. 3. Nill K. 2002. Glossary of Biotechnology Term. 3rd ed. CRC Press LLC, USA. 4. Old RW and Primrose SB. 1994. Principles of Gene Manipulation. Blackwell Scientific Publications, Osney Mead, Oxford, UK. 5. Singleton P and Sainsbury D. 2001. Dictionary of Microbiology and Molecular Biology. 3rd ed. John Wiley & Sons, Ltd. UK.

Chng 1

Cc enzyme dng trong to dng phn t

I. Cac enzyme hn ch Cac enzyme han ch c phn lp t cc sinh vt prokaryote, co kha nng phn huy DNA ca bacteriophage han ch kha nng sinh trng cua chng trong vi khun. Hin nay, ngi ta tm thy hn 900 enzyme han ch khac nhau t khong 250 chng vi sinh vt. Cc enzyme hn ch c ba loi (type): I, II v III. Cc enzyme c dng ph bin hin nay thuc type II, c c ch tc ng n gin nht. y l cc nuclease ct mt v tr c hiu nm bn trong si DNA (ch khng phn hy DNA t hai u), nn c gi l endonuclease. Tn gi y ca chng l cc restriction endonuclease type II, hay c gi n gin l enzyme hn ch (restriction enzyme, RE).

1. Cc enzyme hn ch type II Cch gi tn cc enzyme hn ch da trn cc qui c quc t. Tn chi v tn loi ca sinh vt, m tm thy enzyme, c dng t cho phn u ca tn enzyme (vit nghing) bao gm: ch th nht ca tn chi v hai ch u ca tn loi. V d: enzyme c tch chit t vi khun Escherichia coli th c tn l Eco, cn enzyme c tch chit t vi khun Bacillus globigii th vit l Bgl Ngoi ra, tn gi enzyme hn ch cn c b sung thm phn sau (vit thng), ty thuc vo chng vi khun lin quan v ty thuc vo s c mt hay khng ca cc yu t ngoi nhim sc th. V d: RI trong enzyme EcoRI c ngha nh sau: R l vit tt ca chng RY13, I l bc xc nh u tin trong vi khun (first identified order in bacterium). Gi tr ca enzyme hn ch l tnh c hiu ca chng. Cac enzyme nay ct DNA cac v tr nhn bit ring bit bao gm t 4-6 cp nucleotide co trinh t i xng ao ngc nhau, cac oan ngn nay goi l palindrome (oan i xng: la oan DNA co hai si hoan toan i xng ging ht nhau nu lt ngc u ui). Vi du : enzyme EcoRI nhn bit chui hexanucleotide (6 nucleotide):

Ging nh EcoRI, nhiu enzyme hn ch a tao ra cac oan DNA vi u li (protruding) 5. Mt s enzyme hn ch khc (v d: PstI) tao ra cac oan DNA co u li 5. Trong khi , mt s enzyme hn ch (v du: SmaI) li ct truc i xng tao ra cac oan DNA mang u bng (DNA mach i co hai u si n bng nhau) (Bang 1.1). Bang 1.1. Trinh t nhn bit cua mt s enzyme han ch tiu biu

Vi bn loi nitrogen base trong phn t DNA v gi thit rng trnh t sp xp ca chng l ngu nhin, th tn s mong i (k vng) ca bt k on trnh t xc nh no, theo tnh ton s l 4n, n y l chiu di ca on nhn bit. T , c th thy rng vi cc on c bn nucleotide th c cch 256 cp base chng lp li mt ln, cc on su nucleotide th cch 4096 cp base mi lp li. Tt nhin, cc gi tr ny c th dao ng rt ln, nhng ni chung chiu di ca cc on sinh ra u gn vi cc gi tr tnh ton. Chng hn, mt enzyme nhn bit on trnh t bn nucleotide s sn sinh ra cc on ngn hn so vi on su nucleotide. 2. Gn cac u tn cung c ct bi enzyme hn ch - Cc u dinh (cohesive ends) , cn gi l u li hay u so le. V d: u dnh c to ra nh PstI

- Cc u bng (blunt ends) , cn gi l u th

V d: u bng c to ra nh HaeIII

- Dung enzyme DNA ligase cua phage T4 co th gn lai cac u dinh hoc u bng. Tuy nhin, trng hp u bng thng cho hiu qua thp vi th ngi ta co th dung cac bin phap khac nh: + Gn vo u bng mt oan b sung tao ra u dinh. Vi du: gn oan linker (on ni) bng cch tng hp nhng trnh t oligonucleotide nhn tao c t 10-20 nucleotide lam linker nh sau: 5' NN GAATTC NN 3' 3' NN CTTAAG NN 5' + Dung terminal transferase. Enzyme nay se tao u 3 ca DNA si i mt homopolymer (xem chng 6). 3. Isochizomer Noi chung, cac RE khac nhau nhn bit cac trnh t khac nhau (Bang 1.1), tuy nhin cung co mt s trng hp cho thy co nhng enzyme c phn lp t nhiu ngun khac nhau nhng lai ct trong mt trnh t, cac enzyme o c goi la isochizomer. Hn na, mt vai enzyme nhn bit cac chui tetranucleotide (4 nucleotide) ma trong mt s trng hp cac tetranucleotide nay lai xut hin trong cac chui hexanucleotide cua cac enzyme khac. Vi du: - MboI va Sau3AI cng nhn bit trnh t:

- Trong khi o BamHI nhn bit trnh t:

Trong mt vai trng hp khc cac oan c tao ra nh mt enzyme hn ch nay khi gn vi cac oan c tao ra nh mt enzyme hn ch khac a hinh thanh cac th lai (hybrid) ma cac enzyme b me khng th nhn bit c. Vi du: SalI ct trnh t (GTCGAC) va XhoI ct trnh t (CTCGAG), cac trnh t c sinh ra nay a gn vi

nhau tao thanh th lai ma cac vi tri ct han ch cua chung khng th nhn bit bi cac enzyme SalI va XhoI:

4. Methyl ha S methyl hoa (methylation) nhm mc ch bao v DNA cua cac oan palindrome, nghia la gn gc methyl (CH3) vi tri cn thit nn khng bi RE ct. Vi du: EcoRI nhn bit chui:

Khi co s methyl hoa thi enzyme khng nhn bit c vi tri ct han ch va do o DNA khng bi ct, nhng DNA cua phage khng co gn gc methyl se bi ct bi RE. Trong ky thut gen, enzyme methyl hoa c goi la methylase enzyme. Methylase c dung bao v oan DNA cn gn vao. Tt ca cac chung E. coli u cha hai enzyme methyl hoa DNA la: dam va dcm. - dam (DNA adenine methylase) Enzyme methylase nay gn cac nhom methyl vi tri N6 cua adenine trong chui me 6 5G ATC3 . Nhng DNA ca eukaryote khng bi methyl hoa vi tri N cua adenine. - dcm (DNA cystosine methylase) Enzyme methylase nay gn cac nhom methyl vi tri C5 cua cytosine bn trong cac me me chui 5C CAGG3 hoc 5C CTGG3 . Enzyme ch yu anh hng n s methyl hoa dcm la EcoRII, enzyme BstNI co th nhn bit chinh xac cung mt trnh t nh EcoRII (mc du no ct DNA vi tri xac inh khac trong chui nay). Nu khng th thay BstNI cho EcoRII thi DNA phai c chun bi t cac chung E. coli dcm. 5. Ct DNA bng 5.1. Cc loi m dng trong phn ng ct DNA enzyme hn ch

Mi enzyme hn ch co mt iu kin phan ng ti u, nhit u va thanh phn cua dung dich m la nhng yu t quan trong. Nhit yu cu kha chinh xac con s khac nhau gia cac dung dich m thng la khng ang k. thun tin trong nghin cu ngi ta chia cac enzyme ra lam ba nhom: - Nhom m cao (H). Hoat ng tt nht cac dung dich m co hoat ion cao. - Nhom m trung binh (M). Thich hp vi cac dung dich m co hoat ion trung binh.

- Nhom m thp (L). Thich hp vi cac dung dich m co hoat ion thp. Nh vy, theo cach phn chia nay chi co ba loai m stock la cn chun bi cho phn ng ct DNA bng RE (Bang 1.2). Thng cac m stock trong bang 1.2 c o o pha nng ( 10), va co th gi nhit 4 C/1-2 tun hoc -20 C/khng han inh. Bang 1.2. Cac loai dung dich m cho phan ng ct DNA bng RE

Chu y Ring enzyme SmaI khng th s dung cac loai m trn m cn phi pha dung dich m c bit, bao gm: 20 mM KCl, 10 mM Tris.Cl (pH 8), 10 mM MgCl2, v 1 mM dithiothreitol. 5.2. Phng phap ct DNA bng enzyme hn ch Cac phan ng c trng dung khoang 0,2-1 g DNA/20 L dung dich phn ng hoc it hn. Vi du: a. Pha dung dich DNA bng nc ct hai ln v trung trong eppendorf tube v trung ti dung tich 17 L. b. B sung 2 L m phn ng ( 10) thich hp ca RE va trn u (vortex). V d: BstXI : Dung m cao 10(H) XbaI : 10(H) EcoRI : 10(H) c. B sung 1 unit/L RE va lc u nhe. Mt unit (n vi, k hiu l u) RE c xac inh nh la s lng yu cu u thuy phn hoan toan 1 g DNA plasmid/1 gi dung dich m va nhit thich hp trong 20 L phan ng. d. U nhit thich hp, thi gian tuy thuc vao yu cu. Vi du: BstXI : 1-2 gi/50oC

XbaI : 1 gi/37oC EcoRI : 1 gi/37oC e. Dng phan ng bng cach b sung 0,5 M EDTA (pH 7,5) at ti nng cui cung la 10 mM. - Nu DNA sau khi ct bng RE, c phn tich trc tip trn gel thi b sung 1 L ( 10) gel-loading-dye I, trn u va chay in di. - Nu DNA sau khi ct bng RE, cn c tinh sach thi chit mt ln vi phenol, mt ln vi phenol:chloroform (1:1), mt ln vi chloroform va kt tua DNA bng hai th tich ethanol hoc mt th tich isopropanol. Chu y - Kt qua hoat ng cua enzyme han ch phu thuc rt nhiu vao sach cua DNA. Cac ch phm DNA con ln m chit, phenol hoc EtOH s lam giam cht lng hoat ng cua enzyme. - Enzyme han ch c gi -20oC (hoc -80 o C nu bo qun lu di). Nu ly enzyme ra khoi tu lanh su trong mt thi gian ngn dung thi phai t trn a tuyt (ice bath). II. Cc enzyme 1. DNA polymerase (DNA-dependent DNA polymerase) trng hp

DNA polymerase c dung tng hp DNA trong c th hoc trong iu kin in vitro. 1.1. DNA polymerase I cua E. coli Enzyme nay dich chuyn im t (nick) cua DNA, nick la im t trn mt si cua DNA si i. Chc nng chinh cua enzyme la sa sai doc theo phn t DNA. Nu gp ch sai sot, no se i ngc lai ct bo sau o mi tng hp DNA. DNA polymerase I cua E. coli mang chui polypeptide co khi lng phn t (M) la 109.000 Da vi ba hoat tinh ring bit: - Hot tnh polymerase 53 . DNA polymerase I cua E. coli xc tc cho s tng hp DNA theo chiu 53 bng cch b sung cac gc nucleotide vao u tn cung 3-OH c tao ra khi mt si cua phn t DNA si i bi t. - Hot tnh exonuclease 35. DNA polymerase I cua E. coli ct cc chui nucleotide cc u t do ca DNA, xc tc cho s thoi bin bc thang t u 3 ca c DNA si i v si n khi khng c dNTPs. Trong trng hp c dNTPs, hot tnh exonuclease trn si i s b c ch bi hot tnh polymerase. Trong qu trnh tng hp DNA, hot tnh exonuclease thc hin chc nng c sa (proofreading) bng cch ct b nhng nucleotide lp rp sai.

- Hoat tinh exonuclease 53. DNA polymerase I cua E. coli co th chuyn ch cac nucleotide t phia 5 cua im t va b sung tc thi cac nucleotide t phia 3 tao ra chuyn ng cua im t doc theo DNA.

- ng dung chnh + anh du on DNA lm mu d bng dich chuyn im t. + Khi u cho s tng hp si th hai trong tao dong cDNA. + Xac inh trinh t DNA bng phng phap dideoxynucleotide. 1.2. oan Klenow cua DNA polymerase I cua E. coli Enzyme ny co tac dung lp y cac u khuyt 3 cua DNA si i. Do DNA polymerase I co ba hoat tinh, nhng hoat tinh exonuclease 53 it s dung nn Klenow dung protease ct bt oan co hoat tinh o. Vi vy, khi lng phn t ca enzyme ch con lai 76.000 Da (c gi l oan ln cua DNA polymerase I).

- ng dng chnh + Lm y u khuyt 3 do enzyme hn ch to ra. + anh du u tn cung ca cc on DNA bng cch dng [ a - 32P]dNTPs lm y u khuyt 3. + anh du u tn cng ca cc phn t DNA mang u li 3. u tin, hot tnh exonuclease 35 loi b u li 3 to ra u khuyt 3. Sau , nh s c mt nng cao ca mt tin cht c nh du ng v phng x, s thoi bin bc thang c cn bng do s hp nht ca cc dNTP u 3. + Tng hp si th hai cua cDNA trong to dng cDNA. + Tng hp DNA si i t cc khun mu si n trong pht sinh t bin in vitro. 1.3. DNA polymerase cua bacteriophage T4 (T4-infected E. coli) Enzyme nay ging oan Klenow cua DNA polymerase I cua E. coli . DNA polymerase ca phage T4 (M = 114.000 Da) co hoat tinh polymerase 53 va exonuclease 35 . Tuy nhin, hoat tinh exonuclease cua DNA polymerase phage T4 ln hn cua DNA polymerase I n 200 ln. oan Klenow phi cn co on mi (primer) mi tng hp DNA c, con DNA polymerase phage T4 thi khng cn mi cung tng hp c DNA.

- ng dng chnh + Lm y hoc nh du u khuyt 3 do enzyme hn ch to ra. + anh du u tn cng ca cc phn t DNA mang u li 3, tng t nh ng dng ca on Klenow. + nh du cc on DNA lm mu d. + Bin i u sole ca DNA si i thnh u bng. 1.4. Taq DNA polymerase (Thermus aquaticus) Taq DNA polymerase (Taq pol) la mt loai DNA polymerase chiu nhit (M = 65.000 Da) ca vi khun Thermus aquaticus. Enzyme nay c dung kim tra s c mt hoc khng ca mt gen bng cch xc tc cho s tng hp gen iu kin in vitro nh phan ng chui polymerase (PCR) (xem chng 3). Taq pol thay th cho DNA polymerase I ca E. coli do c kh nng chu nhit cao ph hp vi iu kin phn ng ca PCR, v n c th tng hp mt si DNA di 1 kb trong vng 30 giy 72oC. - Mt trong nhng hn ch ca Taq pol l s chnh xc khng cao trong qu trnh sao chp ca n do b mt c ch c sa (hot tnh exonuclease 35). Enzyme Taq pol thng mi c t l sao chp li 1/10.000 nucleotide, v c th to ra 16% sn phm PCR di 1 kb b t bin trong phn ng khuch i. Mc d c nhc im trn, Taq pol vn c th c dng trong cc th nghim i hi mt trnh t di truyn chnh xc (nh trong to dng phn t). Tuy nhin, kt qu cho ra mt vector mang on chn (sn phm PCR) cn phi c kim tra bng sequencing. - u im ca Taq pol l sn xut cc on gen mang hai u li A. iu ny c bit hu ch trong TA cloning l k thut m vector (plasmid) c s dng

c sn hai u li T b sung vi hai u li A ca sn phm PCR, nh lm tng hiu qu ca phn ng gn.

Ngoi Taq pol, nhiu DNA polymerase chu nhit khc c a ra th trng vi cc chc nng chuyn bit hay hon thin hn. Chng hn: Pfu DNA polymerase (Pfu pol) c tch chit t Pyrococcus furiosus, thng c dng thay cho, hoc kt hp, vi Taq pol. Enzyme ny n nhit hn v c kh nng c sa, v th cho t l sao chp li thp. Hoc Tth DNA polymerase (Tth pol), c tch chit t Thermus thermophilus, c kh nng hot ng nh mt enzyme phin m ngc khi c mt RNA khun mu v ion Mn2+; nhng nu c s hin din ca DNA khun mu v ion Mg 2+, th Tth pol li xc tc phn ng khuch i DNA. Enzyme ny cho php khuch i khun mu l RNA thng qua s hnh thnh cDNA. 2. RNA polymerase (DNA-dependent RNA polymerase) (Bacteriophage SP6-infected Salmonella typhimurium LT2, bacteriophage T7 hoc T3-infected E. coli) Cac bacteriophage SP6, T7 va T3 sn xut enzyme RNA polymerase khi u tng hp RNA trn khun mu DNA si i co mang promoter c trng bacteriophage. Qu trnh tng hp ny khng cn mi.

- ng dng chnh + Sn xut RNA lm mu d. + Xc nh trnh t ca mt phn t DNA c to dng trong mt vector c mang promoter c trng cho bacteriophage SP6, T7 hoc T3.

+ Sn xut mt lng ln RNA t mt phn t DNA c to dng nghin cu v cu trc, iu ha v cc mi tng tc ca phin bn RNA. 3. Enzyme phin m ngc (reverse transcriptase, RNA-dependent DNA polymerase) y la enzyme tng hp DNA t khun mu RNA. Enzyme nay co trong cac virus: AMV (avian myeloblastosis virus), Mo-MLV (moloney murine leukemia virus) thuc nhom virus ngc (retrovirus: virus mang RNA) va co cac hoat tinh sau: - Hoat tinh polymerase 53. Tng hp DNA theo chiu 53. C cht cua no la RNA hay DNA (si n hoc si i):

- Hoat tinh RNase H. Bao gm hai hoat tinh exoribonuclease 53 va 3 5 phn hy cac DNA hoc RNA trong t hp lai.

Enzyme nay c dung trong ky thut di truyn la nh vao kha nng tng hp c cDNA ca chng (xem chng 6). Ngi ta co th tch chit mRNA nhng t bao tng hp protein manh, sau o dung enzyme reverse transcriptase tng hp cDNA. Co th tng hp nn oligonucleotide ri dung no xac inh trinh t cua DNA. 4. Terminal transferase

(Tuyn c ca b) Hoat tinh cua enzyme nay la xc tc gn cc nucleotide vo u 3-OH t do ca DNA si i lam li ra mt u cui si DNA. Phn ng gn ny l ngu nhin, do thnh phn nucleotide ca u li ph thuc vo nng ca bn loi nucleotide c trong phn ng.

- ng dng chnh + Thm ui ng trng hp (homopolymer) to ra u so le cho phn t DNA dng trong to dng (xem chng 6). + nh du u 3-OH ca DNA trong k thut xc nh trnh t gen theo Maxam v Gilbert. III. Cc enzyme gn Enzyme ligase thng dung la cua phage T4 xm nhim trong E. coli. Chc nng cua enzyme ny la ni cac oan h bng lin kt cng hoa tri, s dung nng lng ATP. 1. Bacteriophage T4 DNA ligase (Bacteriophage T4-infected E. coli) Enzyme nay l mt chui polypeptide (M = 68.000 Da) xuc tac cho s tao thanh lin kt phosphodiester gia u 3-OH t do ca mt phn t DNA ny vi u cui 5-PO4 ca mt phn t DNA khc. DNA ligase c tc dng cho c trng hp u so le ln u bng, tuy nhin i vi u bng enzyme i hi nng cao hn v iu kin phn ng cng khc.

2. Bacteriophage T4 RNA ligase (Bacteriophage T4-infected E. coli) Enzyme ny xuc tac cho mi lin kt cng hoa tri gia nhm 5-PO4 ca DNA si n hoc RNA vi nhm 3-OH ca DNA si n hoc RNA. Do c cht thch hp cho enzyme ny l cc phn t nh nn n thng c dng nh du u 3 ca cc phn t RNA s dng lm mu d.

3. Bacteriophage T4 polynucleotide kinase (Bacteriophage T4-infected E. coli) Enzyme bacteriophage T4 polynucleotide kinase xc tc chuyn nhm -phosphate ca ATP ti u 5 ca DNA hoc RNA. Hai loi phn ng thng c th xy ra l phn ng thun v phn ng trao i. Trong phn ng thun, nhm -phosphate c chuyn ti u 5 ca DNA c dephosphoryl ha (mt nhm phosphate). Trong

phn ng trao i, khi c mt ADP tha, enzyme s chuyn nhm 5 phosphate t DNA c phosphoryl ha ti ADP, DNA sau s c phosphoryl ha tr li bng cch nhn mt nhm -phosphate c nh du ng v phng x t mt phn t [-32P]ATP. - ng dng chnh + nh du phng x u 5 ca DNA phn tch trnh t bng phng php Maxam v Gilbert (1977), dng lm mu d trong cc k thut lai phn t (xem chng 5). + Phosphoryl ha cc on ni (linker) v cc on DNA khng c nhm 5 phosphate chun b cho phn ng gn trong phng php to dng.

4. Alkaline phosphatase (E. coli v rut b) C hai enzyme alkaline ca vi khun (bacteria alkaline phosphatase, BAP) v rut b (calf intestinal alkaline phosphatase, CIP) u xc tc loi b nhm 5 phosphate khi DNA v RNA.

- ng dng chnh + Loi b nhm 5 phosphate khi DNA hoc RNA trc khi nh du u 5 bng 32P. + Loi b nhm 5 phosphate khi cc on DNA ngn cn s t gn. Trong k thut to dng, khi mt vector c m vng DNA bng mt RE ri sau c loi b nhm 5 phosphate th n khng th t gn li (t ti to li vng). Ch khi c on DNA ngoi lai mang cc u 5 phosphate cn thit c a vo th phn ng gn gia vector v DNA ngoi lai mi xy ra. IV. Cc enzyme phn ct L nhm cc enzyme xc tc thy phn lin kt phosphodiester trong phn t DNA hoc RNA, bao gm cc endonuclease (ct bn trong phn t nucleic acid) v exonuclease (ct bn ngoi phn t nucleic acid). Cc RE cng l cc endonuclease, nhng chng c tnh c hiu rt cao cho tng trnh t 4 hoc 6 nucleotide. Di y l cc nuclease chnh thng c s dng: 1. Deoxyribonuclease I (DNase I) (Ty b) DNase I xc tc thy phn DNA si n hoc si i cc lin kt phosphodiester nm bn cnh cc pyrimidine nucleotide, to ra cc oligonucleotide v cc oligonucleotide c u tn cng 5' monophosphate. Khi c mt Mg2+, DNase I tc dng c lp trn mi si DNA, v cc v tr ct c phn b ngu nhin.

Khi c mt Mn2+, DNase I s ct c hai si DNA gn nh cng mt v tr to ra cc on DNA u bng hoc u li nhng ch nh ra mt hoc hai nucleotide.

- ng dng chnh + To ra cc im t (nick) trn DNA si i nh du mu d ng v phng x bng phng php dch chuyn im t. + To ra cc dng ngu nhin phn tch trnh t trong bacteriophage M13 vector. + Phn tch phc hp protein:DNA (DNase footprinting). + Loi b DNA trong cc phn on RNA hay protein. 2. Nuclease S1 (Aspergillus oryzae) Enzyme nuclease S1 ct DNA si n hoc RNA tao ra u 5 monophosphate hoc cac oligonucleotide. DNA si i va th lai DNA:RNA it chiu tac dung cua enzyme nay. Tuy nhin, cac nucleic acid si i se bi nuclease S1 ct hoan toan nu chung bi x ly vi mt lng rt ln enzyme. Mt lng va u cua enzyme se tach cac nucleic acid si i cac im t hoc cac l hng nho thanh hai hoc nhiu oan.

- ng dng chnh + Phn tich cu truc th lai DNA:RNA. + Loai bo cac phn si n trn u sole ra khoi oan DNA si i tao ra cac u bng. + M vong cp toc xut hin trong qua trinh tng hp cDNA si i.

Mt enzyme c hot tnh tng t nuclease S1 l mung-bean nuclease (endonuclease) c tch chit t mm u xanh (Phaseolus aureus). 3. Exonuclease III (Exo III) (E. coli) Exo III xc tc loi b cc 5 mononucleotide t u 3 OH ca DNA si i (DNA mch thng hoc mch vng cha cc im t hoc l hng). Hot tnh enzyme cho kt qu to thnh cc vng si n di trong DNA si i. Enzyme ny c 3 hot tnh khc nhau: endonuclease c hiu cho apurinic DNA, RNase H v 3 phosphatase (loi b u 3 phosphate nhng khng ct cc lin kt phosphodiester bn trong). Exo III khng ct cc DNA si n hoc DNA si i c u li 3.

- ng dng chnh + To ra cc cu trc si n mt s vng trn phn t DNA dng lm c cht cho on Klenow ca DNA polymerase I ( sn xut mu d c trng cho tng si). + To ra cc t bin khuyt on (deletion) ti cc trnh t tn cng ca DNA si i mch thng. Phn ng ny thng phi hp vi mung-bean nuclease hoc nuclease S1. Mt exonuclease tng t l nuclease BAL31, c hot tnh exonuclease 53 v 35, c kh nng to cc DNA si i u bng m khng cn s hin din ca nuclease S1.

4. Ribonuclease (RNase A) (Ty b) Enzyme xc tc thy phn RNA thnh cc on nh hn. RNase A c hot tnh rt mnh, hin din mi ni v rt bn vng (khng b mt hot tnh khi b x l 90C trong 1 gi). RNase A ct lin kt phosphodiester nm ngay sau mt pyrimidine ca mt RNA si n.

- ng dng chnh + Loi b RNA trong cc ch phm DNA hay protein. + Loi b cc vng khng bt cp trn RNA trong th lai RNA:DNA. 5. RNase H Enzyme RNase H l mt loi ribonuclease c kh nng ct lin kt 3-O-P ca RNA trong si i ca th lai DNA:RNA to ra cc sn phm c u tn cng 3OH v 5-PO4. RNase H l mt endonuclease khng c hiu, xc tc ct RNA thng qua c ch thy phn nh mt ion kim loi ha tr 2 lin kt vi enzyme. Trong to dng phn t, RNase H xc tc ct c hiu RNA trong th li RNA:DNA m khng ct DNA hoc RNA khng trong th lai, enzyme ny thng c dng ph hy khun mu RNA sau khi tng hp si cDNA th nht bng phin m ngc, tip tc tng hp si cDNA th hai to thnh mt si i cDNA. V. Cac protein lin kt DNA si n Cac protein lin kt DNA si n (single stranded DNA-binding proteins, SSB) kt hp vi DNA si n nhng khng kt hp vi DNA si i. Chung c xem nh la cac cht phan ng trong tao dong phn t xut phat t ch chung co kha nng pha v s n inh cua cac cu truc th cp bn trong si nucleotide; tng nhanh s tai u cua cac polynucleotide b sung va tng hoat tinh cua cac enzyme DNA polymerase bng cach loai bo cac cu truc th cp bn trong si la nhng yu t ngn can s tin trin cua cac enzyme nay. Nh tinh cht nay, cac protein SSB tr thanh cac cht phan ng hu ich trong xac inh trinh t DNA.

- ng dng chnh + Phat sinh t bin im trc tip bao gm D-loops. + Tng chiu dai chui cua san phm trong moi phan ng c xuc tac bi DNA polymerase trn cac khun mu dai. Cac protein SSB c bit co th kich thich cac DNA polymerase c hiu dung trong cac phan ng phn tich trinh t chui DNA. Tai liu tham kho/c thm
1. H Hunh Thy Dng. 1998. Sinh hc phn t. NXB Gio dc, H Ni. 2. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA and Struhl K. 2002. Short Protocol in Molecular Biology. Vol 1 and 2. 5th ed. John Wiley & Sons, Inc. USA. 3. Brown TA. 2001. Gene Cloning-An Introduction. 4th ed. Blackwell Science, Oxford, UK. 4. Glick BR and Pasternak JJ. 2003. Molecular Biotechnology: Principles and Applications of Recombinant DNA. 3rd ed. ASM Press, USA. 5. Maniatis T, Fritsch EF and Sambrook J. 1989. Molecular Cloning-A Laboratory Manual. Cold Spring Habor Laboratory Press, USA. 6. Ohman DE. 1989. Experiments in Gene Manipulation. Prentice Hall, Englewood Cliffs, New Jersey, USA. 7. Primrose SB, Twyman R and Old RW. 2001. Principles of Gene Manipulation. 6th ed. Blackwell Science, Oxford, UK.

Chng 2

in di gel
I. Nguyn l chung in di la ky thut c dng phn tch v mt i khi tinh sch cac i phn t-c bit l cc protein v cac nucleic acid-trn c s kch thc/khi lng, in tich v cu hnh cua chung. Khi cc phn t tch in c t trong mt in trng, chng s dch chuyn hng n cc dng (+) hoc cc m (-) ty theo in tch ca chng. Ngc vi protein, loi phn t c in tch thc hoc dng hoc m, cc nucleic acid c mt in tch m khng i nh khung phosphate ca mnh, v v th ch dch chuyn hng n cc dng. Cc phn t protein v nucleic acid c th c chy in di trn mt khun (support matrix) nh giy, cellulose acetate, gel tinh bt, agarose hoc polyacrylamide

gel. Trong gel ca agarose v polyacrylamide c s dng ph bin nht. Thng thng, gel l mt khun c dng phin mng c cc ging np (loading) mu. Gel c ngm trong m in di cung cp cc ion dn truyn dng in v mt vi loi m duy tr pH mt gi tr khng i tng i. II. in di agarose gel Agarose (polysaccharide) c khi lng phn t xp x 120.000 Da (Hnh 2.1) l mt trong hai thnh phn chnh ca agar1 chim khong 70%, phn kia l agaropectin chim khong 30%. Agarose l mt polymer mch thng khng b sulphate ha cha hai gc xen k nhau l D-galactose v 3,6-anhydro-L-galactose. Agarose gel l mt cht trong sut (transparent) hoc trong m (transluent) ging nh agar, c to thnh khi hn hp agarose v nc (hoc m in di) c un nng ti >100oC v sau c lm lnh; dng gel xut hin khong 40-45oC. Agarose gel c ng dng rng ri lm gi th cho cc nucleic acid trong k thut in di ngang (horizontal electrophoresis) hoc lm gi th cho mi trng nui cy bacteriophage (top agarose).

Hnh 2.1. Cu trc phn t ca agarose. n v agarobiose (v d: hai phn t ng) l mt monomer trong agarose polymer. C khong 400 monomer trn mt chui polymer.

Cac phn t nucleic acid co khi lng va in tich khac nhau c tach ra khi di chuyn t cc m sang cc dng cua h in di trong mt in trng co in th va cng thich hp. Ky thut nay n gian va thc hin nhanh. Hn na, vi tri cua DNA trong gel c xac inh trc tip: cac bng DNA trong gel c nhum nng thp cua thuc nhum huynh quang ethidium bromide (EtBr) v co th pht hin di anh sang t ngoai (ultraviolet-UV). in di agarose gel c s dng trong cc trng hp sau: - c lng kch thc ca cc phn t DNA sau khi thc hin phn ng ct hn ch (v d: lp bn hn ch ca DNA c to dng). - Phn tch cc sn phm PCR (v d: trong chn on di truyn phn t hoc in du di truyn).

- Phn tch DNA h gen c ct hn ch trc khi thm tch Southern, hoc RNA trc khi thm tch Northern. u im ca phng php ny l gel c rt d dng, khng gy bin tnh mu, v bn vng vt l hn polyacrylamide. Mu cng d thu hi. Nhc im l agarose gel c th b nng chy trong qu trnh in di, m c th b tiu hao, v cc dng khc nhau ca nucleic acid c th chy khng n nh. 1. Cac yu t anh hng n tc dich chuyn in di trong agarose gel 1.1. Kich thc cua phn t Cac phn t DNA mach thng si i i qua ban gel cac tc ty l nghich vi ham log10 cua khi lng phn t cua chung (Hinh 2.2). Do , cc phn t DNA c kch thc cng ln (khi lng phn t ln) th tc dch chuyn cng chm.

Hinh 2.2. Mi quan h gia kich thc DNA va linh ng in di cua no

1.2. Nng agarose oan DNA mang kich thc nht inh s dich chuyn cac tc khac nhau qua cac ban gel cha cac nng agarose khac nhau. Mi quan h tuyn tinh gia ham logarithm cua linh ng in di cua DNA ( ) va nng gel ( ) c biu din bng biu thc:

Trong

o: linh ng in di t do.
Kr: h s tr hon in di (retardation), c thit lp thng qua mi lin quan gia cac tinh cht cua gel vi hinh dang va kich thc cua cac phn t dich chuyn.

Nh vy, dung gel cac nng khac nhau co th phn tch c cac oan DNA co kich thc khac nhau (Bang 2.1). Nng agarose cao c kh nng phn tch cc on DNA nh, trong khi nng agarose thp li cho php phn tch cc on DNA ln hn.

Bang 2.1. Cac thng s in di DNA bng agarose gel

Hnh 2.3 minh ha s dch chuyn ca tp hp cc on DNA trong hai mu ba nng khc nhau ca agarose, tt c chng trong mt khay gel v c in di cng mt in p (voltage) trong mt thi gian xc nh. Kt qu cho thy, cc on ln c phn tch tt hn gel 1%, trong khi cc on nh thch hp vi gel 2%.

Hnh 2.3. in di cc mu DNA ging nhau cc nng agarose khc nhau. A: 1%, B: 1,5% v C: 2%.

1.3. Cu hinh cua DNA Cac DNA dang vong ong (form-I), vong t (form-II) va mach thng (form-III) co cung mt khi lng phn t se dich chuyn trn agarose gel cac tc khac nhau. Nhn chung, DNA ca cc plasmid mch vng dch chuyn nhanh hn DNA ca plasmid cng loi nhng c dng mch thng. Hu ht plasmid mch vng cha t nht hai dng DNA c cu trc khng gian khc nhau l dng vng ng (siu xon) v vng t. Hnh 2.4 minh ha kt qu in di vi plasmid mch vng bn tri v plasmid cng loi mch thng bn phi.

Hnh 2.4. in di cc plasmid c cu hnh khc nhau

1.4. Thanh phn base va nhit

Tp tinh in di cua DNA trong agarose gel khng bi anh hng ro rt bi thanh phn base cua DNA hoc nhit ma o gel c chay. Trong agarose gel, tinh linh ng in di tng i cua cac oan DNA co kich thc khac nhau khng thay i trong o o khong t 4 C n 30 C. Noi chung, agarose gel thng c chay nhit phong.

2. Phng phap in di agarose gel 2.1. m pH Mt s m in di thich hp cho DNA si i thng c dng la Tris-acetate EDTA (TAE), Tris-borate-EDTA (TBE) v Tris-phosphate-EDTA (TPE) nng khoang 50 mM va pH 7,5-7,8 (Bng 2.2). Thng do thoi quen, TAE c s dung nhiu nht, nhng kha nng m cua no lai thp. m TBE v TPE u co kha nng hoa tan tt cac oan DNA va co kha nng m cao hn mt cach ro rt. Cc on DNA s dch chuyn vi cc tc hi khc nhau mt cht trong ba loi m trn do s khc nhau v cng lc ion ca m. m khng nhng thit lp mt gi tr pH, m cn cung cp cc ion h tr cho dn (conductivity). Nu chng ta dng nc thay v l m trong qu trnh in di, th s khng c s dch chuyn cn thit ca DNA trong gel. Ngc li, nu s dng m nng m c (v d: dung dch stock 10), th nhit trong gel c th tng cao v lm nng chy n.

2.2. Chun bi agarose gel Co nhiu loai agarose khac nhau thich hp chay in di, loai agarose dung tt nht trong thi nghim la type-II-agarose co ni thm thu thp (low-endo-osmotic). No d dang chay ra va tao thanh mt dung dich trong sut, kt qua la gel an hi thm chi cac nng thp. Tuy nhin, type-II-agarose d bi bn bi sulphate polysaccharides (SP), hn na SP con c ch cac enzyme nh ligase, polymerase va RE. Vi th, cac oan DNA dung ly t nhng gel nh th phai c lam sach cn thn trc khi chung c dung nh la cac khun mu hoc c cht cho cac enzyme nay. Bang 2.2. Cac m s dung ph bin trong in di

2.3. Nhum DNA trong agarose gel Phng phap quan sat DNA trong agarose gel thun li nht la dung thuc nhum huynh quang EtBr. EtBr c dung pht hin DNA si i va RNA si n. Tuy nhin, ai lc (affinity) cua thuc nhum i vi nucleic acid si n la tng i thp va co hiu sut huynh quang khng cao. Sau khi nhum, EtBr s xen vo gia cc base ca nucleic acid v cho php pht hin d dng chng trong gel. Thng EtBr (0,5 g/mL) c a vao trong agarose gel phn ng nhum xy ra trong sut qu trnh in di. Mc du tinh linh ng in di cua DNA si i mach thng giam khi co mt thuc nhum (khoang 15%), nhng kha nng xac inh gel trc tip di anh sang UV trong sut qua trinh in di hoc giai oan cui co u th ro rt. Tuy nhin, nu cn co th chy in di gel khng co EtBr va chi nhum DNA hoc RNA sau khi in di xong. Gel c ngm trong m in di hoc H 2O cha EtBr (0,5 g/mL)/45 phut nhit phong.
Chu y Ethidium bromide la mt tac nhn gy t bin manh. V th, lun lun eo gng tay khi cm gel hoc dung dich cha thuc nhum.

2.4. Thu hi DNA t agarose gel

Nhiu phng phap a c xy d ng thu hi DNA t agarose gel, nhng khng co phng phap nao la hoan toan ti u. Co hai vn chinh: th nht a s cac loai agarose u bi nhim bn b i sulphate polysaccharides, cac san phm sau o c chit t gel cung v i DNA, chung la nhn t c ch co hot tnh cua nhiu loai enzyme (RE, ligase, kinase, polymerase) dung trong cac b c tao dong tip theo sau; th hai hiu sut chit DNA t agarose gel phu thuc vao khi lng phn t cua no, cac oan DNA co chiu dai <1 kb co th c thu hi hon ton, khi khi lng phn t cua DNA tng thi hiu sut chit giam, cac oan DNA co kich th c >20 kb c thu hi kem (khoang hn 20% san l ng). C nhiu phng php thu hi v tinh sch DNA t agarose gel, di y l mt vi phng php chnh:

2.4.1. Dng agarose c nhit nng chy thp (low melting temperature) Mu agarose gel c nhit nng chy thp cha on DNA quan tm c ct ra khi bn gel v cho vo trong m vi t l 1:1, b sung mui n nng 0,5 M, sau c nng chy 70oC ha tan hon ton trong m. Dung dch gel c DNA c chit bng phenol/chloroform v kt ta. Lu DNA c tch chit bng phng php ny khng thch hp cho mi phng thc thao tc tip theo, do s hin din ca cc nhn t c ch trong agarose.

2.4.2. Ly tm Mu agarose gel c cha bng DNA quan tm c ct ra v cho vo m chit, lm nng ha tan hon ton, sau cho dung dch gel ln trn mt lp (mng) bng thy tinh silicon ha t trong mt ct lc nh loi 0,5 mL (cn gi l spin column). Ct ny c cho vo mt tube vi ly tm loi 1,5 mL v c ly tm tc cao 10.000-15.000 rpm trong 10-15 pht. DNA s lin kt vi mng cn dung dch m s i qua lp bng thy tinh vo tube vi ly tm. Cho m ra vo ct v ra ct vi ln bng cch ly tm nhanh. Thay tube vi ly tm mi, cho m ha tan DNA vo ct v ly tm dung ly (elution) DNA ra khi mng vo tube. Dung dch DNA thu c c th c tinh sch thm nu cn. Cng c th ng lnh mu gel trc khi dng phng php ly tm ci thin hiu sut thu hi DNA.

2.4.3. in di vo by Mt ci rnh nh c ct trong agarose gel ngay pha trc ca bng DNA quan tm. Rnh ny c lm y bng glycerol v gel c in di nhanh tr li

chuyn DNA t gel vo trong dung dch glycerol (qu trnh in di c th c kim sot bng UV). Dung dch glycerol-DNA sau c chit bng pipette. Hoc sau khi in di, mt khe nh c ct trong gel ngay pha trc bng DNA quan tm v t trong khe mt mu giy loi NA-45. Gel sau c in di tr li v bng DNA quan tm s dch chuyn vo trong mu giy. Mu giy sau c ra v DNA c dung ly trong m bng cch un nng mu giy ti 70oC. DNA sau c th c tch chit bng phenol v kt ta.

2.4.4. Dng ht thy tinh Mu gel quan tm c ha tan trong dung dch mui NaI (mt loi chaotropic salt) nng khong 4 M. Cc ht thy tinh sau c b sung vo dung dch lin kt hiu qu vi cc on DNA c phng thch nng cho ca NaI. RNA, protein v cc tp cht khc khng lin kt vi cc ht thy tinh. Tip theo l cc chu k ra v kt ta tiu th, DNA tinh sch c dung ly khi ht thy tinh trong m mui thp. Tuy nhin, phng php ny mc d nhanh v hiu qu nhng li c phm vi ti u hp. Thu hi cc on DNA nh (500-800 bp) l khng hiu qu lm v cc on ln (>15 kb) c th lin kt vi cc ht thy tinh khc nhau v cc si DNA c th b ph v trong sut cc chu k ra.

3. Quy trinh in di 3.1. Chun bi agarose gel Cho 1% type-II-agarose vao 100 mL m 0,5 TAE (nu khun gel ln thi tng theo ty l trn). un trong ni kh trng hoc l vi sng cho n khi agarose tan hoan toan. Nu qua trinh un mt nc qua nhiu thi phai thm nc cho u 100 mL. o ngui khoang 60 C va vao bung in di co cai sn lc. Chiu day gel khoang 5 mm la thich hp. Sau 30-40 phut, khi gel a ng cng, co th g lc ra.

3.2. t mu DNA vao ging Tuy thuc vao tng muc ich in di khac nhau co th s dung nng DNA cao hoc thp. Chng han theo ty l sau:
DNA (1 g/1 L) m mau bromophenol (10 loading dye) Nc ct 2 ln Tng s 1 L 1 L 9 L 11 L

Dung micropipette hut 11 L dung dich trn cho vao ging. Lu mu chay in di phai co dung tich th tich cua ging. Thng t mu sau khi a dich m 0,5 TAE vao bung in di cho ngp gel, it khi t mu trc ri m sau (nap kh). d lam vic nn dung micropipette loi 20-200 L va t bung in di trn ban co nn en. Khi tng in p ca qu trnh in di, cc on DNA ln thng dch chuyn nhanh hn so vi cc on nh. Tuy nhin, c phn gii (resolution) tt nht i vi cc on DNA c kch thc ln hn 2 kb th in p c s dng phi nh hn 5 V/cm (gi tr cm c tinh theo khoang cach gia hai in cc ch khng phi l chiu di ca gel). DNA dch chuyn t cc m n cc dng. Quan sat s dch chuyn bng mau bromophenol bit luc nao cn ngng in di (Hnh 2.5).

3.3. Nhum DNA bng EtBr Sau khi chay in di xong ly nhe ban gel ra khoi khun va ngm vao dung dich EtBr nng 0,5 g/mL, trong thi gian 30-45 phut, tt nht trn mt may lc nhe ( 10 vong/ phut ). dich nhum EtBr vao mt binh ring x ly va ra ban gel bng cach ngm trong nc ct hai ln, mi ln 15-20 phut. EtBr thng pha sn dang m c 5 mg/mL o va gi 4 C trong ti.

Hnh 2.5. S minh ha cc bc trong qu trnh in di agarose gel

3.4. Quan sat va chup anh Ban gel sau khi nhum EtBr c quan sat di anh sang t ngoai ( = 302 nm) (Hnh 2.6). Dung thit bi chuyn dung phn tch v lu tr hnh nh DNA (Gel Documentation System).

Chu y Khng nhin vao anh sang t ngoai nu khng co kinh loc thich hp.

Hnh 2.6. Hnh nh in di DNA chy trn agarose gel 1%, 3 Volts/cm v nhum bng EtBr. Cc bng DNA c mu sng. SM: Chun kch thc ca DNA (1 kb ladder). Cc ng 1, 2 v 3: mu plasmid DNA c ct bng 3 enzyme hn ch khc nhau.

III. in di polyacrylamide gel Acrylamide la mt monomer co cu truc nh sau:

Khi co mt cac gc t do, c cung cp bi ammonium persulphate va n inh b i TEMED (N,N,N,N-tetramethylethylene-diamine), phan ng chui c bt u trong o cac acrylamide monomer c polymer hoa (trung h p) thanh mt chui dai. Khi bisacrylamide (N,N-methylenebisacrylamide) c b sung trong phan ng polymer hoa, cac chui se lin kt cheo (cross-linking) tao thanh dang gel. xp cua gel c xac inh bi chiu dai cua chui va m c lin kt cheo. Chiu dai cua chui polyacrylamide c xac inh bng nng acrylamide trong phan ng polymer hoa (gia 3,5% va 20%). Ngoi protein, polyacrylamide gel cng c dng in di DNA (k c RNA v DNA/RNA). Gel phai c rot vao gia hai tm kinh (gel plates) c ngn cach bi cac ming m (gel spacers) (Hnh 2.7). Hu ht cac dung dich acrylamide c ngn khng tip xuc vi khng khi han ch s c ch trung hp gy ra bi oxygen. Gel co th dai t 10-100 cm tuy thuc vao yu cu phn tich va chung thng c chay trong phng thng ng.

Hnh 2.7. S v hnh nh ca bung in di polyacrylamide gel

Phm vi kch thc phn t v phn gii ca qu trnh phn tch ty thuc vo nng ca acrylamide v bis-acrylamide (v t l ca chng) do chng to ra khun gel vi cc phn trm khc nhau ca acrylamide v bc ca lin kt cho. Nng thp to ra cc l c kch thc ln hn v v th c th phn tch cc phn t ln hn. Lin kt cho ca cc monomer v cc tc nhn lin kt c th c iu chnh kim sot mc xp ca khun gel. iu ny cho php ti u ha mt khun gel phn tch mt phm vi kch thc c bit ca cc phn t protein. 1. in di nucleic acid in di polyacrylamide gel c dung phn tch va thu hi cac oan DNA co chiu dai t 5-2.000 bp. Nng polyacrylamide gel c s dng trong kho ng t 3,5-20% tuy thuc vao kich th c cua oan DNA quan tm. Hai loai polyacrylamide gel thng c s dung la: - Polyacrylamide gel khng bin tinh dung phn tch va tinh sach cac oan DNA si i. - Polyacrylamide gel bin tinh bng urea v formamide (sequencing gel) dung phn tch va tinh sach cac oan DNA si n. Phn ny ch gii thiu polyacrylamide gel khng bin tnh l loi gel hay c s dng nht. Hiu qua cua s phn tch DNA trong loi gel ny phu thuc vo nng cua acrylamide (Bang 2.3), kch thc v in tch ca DNA.
Bang 2.3. Hiu qua cua s phn tch DNA trong polyacrylamide gel khng bin tinh

1.1. Chun bi polyacrylamide gel khng bin tinh Kich thc ph bin cua tm kinh gel th ng la 20 cm 40 cm. Ming m day khoang 0,5 mm-2,0 mm. Cac gel day hn th ng nong ln trong qua trinh in di nn se lam nhoe cac bng DNA, vi th ng i ta th ng s dung cac gel mong. Tuy nhin, khi khi chay mt l ng l n DNA (>1 g/bng) thi cn chun bi gel day. D i y m ta cc bc chun bi va s dung polyacrylamide gel khng bin tinh:

1.1.1. Chun bi cac dung dich sau: - 30% acrylamide (bao gm bis-acrylamide) - 1 TBE - 10% ammonium persulphate

1.1.2. Lau sach hai tm kinh dung lam khun gel va ming m bng KOH/methanol hoc ethanol. X ly b mt cua hai tm kinh bng dung dich silicon ngn gel dinh cht vao hai tm kinh gy rach gel lc ly no ra khoi khun sau khi in di xong.

1.1.3. Tinh toan th tich cua cac dung dich dung tao polyacrylamide gel trn c s kich thc tm kinh, day cua ming m (xem bang 2.4). 1.1.4. B sung 35 L TEMED vao mi 100 mL dung dich tao polyacrylamide gel, trn hn hp bng cach vortex. Rot dung dich tao gel vao gia hai tm kinh. Gn nhanh lc

vao, tranh bot khi rng lc (ging). inh cua rng lc phai hi cao hn mep gel. Nu cn, b sung thm mt it dung dich tao gel lam y mep gel.

Bang 2.4. Dung tich cua cac cht dung cho polyacrylamide gel

1.1.5. Cho phep acrylamide polymer hoa trong khoang 60 phut nhit phong, b sung thm dung dich tao gel nu thy gel co vao nhiu.

1.1.6. Sau khi polymer hoa hoan toan, rt lc cn thn, thao bng dinh ra khoi ay khun gel. Gn khun gel vao bung in di va lam y bung in di bng m 1 TBE, dung Pasteur pipette pha cac bot khi ra khoi ay gel va cac ging bng m 1 TBE.

1.1.7. Trn cac mu DNA vi mt lng thich hp cua m 6 gel-loading dye. t mu vao trong ging bng micropipette hoc Hamilton syringe.

1.8. Ni bung in di vi b ngun. Polyacrylamide gel khng bin tinh thng chay in di trong khoang 1 V/cm n 8 V/cm. Chay gel cho n khi thuc nhum chi thi dich chuyn n vi tri mong mun. Tt ngun, ly khun gel ra va t ln ban. Thao tm kinh nho mt trc ra, tm kinh ln phia sau c dung lam gia , va chun bi nhum gel.

1.2. Phat hin DNA trong polyacrylamide gel 1.2.1. Nhum bng ethidium bromide Polyacrylamide co th lam mt huynh quang cua EtBr, do o khng th phat hin cac bng DNA bng phng phap nay nu ham lng cua no thp hn 10 ng. Ngm nhe gel cung vi tm kinh vao trong dung dich nhum (0,5 g/mL EtBr trong 1 TBE). Cho va u dung dich nhum phu hoan toan ln b mt gel. Sau khi nhum 30-45 phut nhit phong, ly gel va tm kinh ra, cn thn thm kh b mt gel bng giy thm Kimwipe. Phu ln b mt gel bng giy nylon (Saran wrap), tranh tao ra bot khi hoc np gp cua Saran wrap. Sau lt ngc gel va t no trn may soi t ngoai ultraviolet transilluminator (Gel Documentation System), ly tm kinh ra tin hanh chup anh.

1.2.2. Nhum bng thuc nhum bc Thuc nhum bc c s dng t nhng nm 1980, cho php pht hin mt lng protein rt nh dng vt trong polyacrylamide gel. Sau , loi thuc nhum ny c hon thin v m rng phm vi ng dng cho cc phn t nucleic acid. Hai phng php nhum bc thng c s dng l: 1) Dng cc dung dch diamine silver hoc ammonical silver cho thm vo gel v pha long cc dung dch acid ca formaldehyde pht trin hnh nh. 2) Thm dung dch silver nitrate vo gel trong mi trng acid yu v dng formaldehyde kh c chn lc cc ion bc thnh bc kim loi di iu kin kim. Nhn chung, phng php diamine kim km nhy hn nhng thch hp i vi cc gel dy, trong khi phng php acid nhanh v bt mu tt hn i vi cc gel mng. Thuc nhum bc c s dng hiu qu pht hin mt lng nh (picogram, pg) nucleic acid. Gii hn pht hin DNA si i khi quan st bng mt thng l vo khong 1 pg/mm2 mt ct ngang ca bng DNA, nhy gp 1.000-10.000 ln phng php nhum bng EtBr. Mc pht hin ny c th so snh vi phng php nh du ng v phng x hoc hunh quang. Qu trnh nhum bao gm cc bc sau: - C nh nucleic acid trong acetic acid 7,5% ti thiu 5 pht ngn cn s khuch tn ca cc phn t nucleic acid c phn tch trong gel, loi b v trung ha cc ha cht khng mong mun (urea v m). - Ra gel trong nc kh ion ti thiu 2 pht loi b acetic acid, cc cht bn dng vt v phn tha ca cc thnh phn gel ha tan sau khi c nh. - Nhum gel trong dung dch bc vi s c mt ca formaldehyde ci thin nhy v tng phn. Thi gian nhum ti u khong 20 pht. Tuy nhin, i

vi loi gel c tm polyester (810 cm) th ch cn 10 pht l c cht lng hnh nh cao m khng lm gim nhy. - Ra gel sau khi nhum bc loi b thuc nhum tha c th gy ra kt ta mu nu trong qu trnh pht trin mu. Thng thng, c th dng dung dch pht trin mu ra gel. - Pht trin mu ca gel bng hn hp dung dch sodium carbonate (4 g/L), sodium thiosulfate (4 M) v formaldehyde (khong 0,028-0,111%) gim cc background khng c hiu mt cch hiu qu. Nhit ra thch hp t 8-10 oC, thi gian ra thay i ty thuc vo thnh phn ca dung dch pht trin mu trong khong t vi giy n vi pht. - Dng phn ng pht trin mu khi thu c hnh nh ti u cng nhanh cng tt bng cch dng acetic acid lnh (4oC) 7,5%.

1.2.3. Phong xa t ghi + Khng c inh, lam m gel Nu mun thu hi bng DNA co hoat tinh phong xa t gel thi gel phai c c inh hoc lam kh. Nu ch mun quan st kt qu in di th c th tin hnh nhanh nh sau: - Goi ban gel cung vi tm kinh trong giy nylon Saran wrap. Tranh tao bot khi hoc np gp cua Saran wrap. anh du bng mc phong xa ln b mt Saran wrap. - Lt ngc gel va u v i phim X-quang (tin hanh trong phong ti) -70oC trong khoang th i gian thich h p. + C inh, lam kh gel Cac polyacrylamide gel cha DNA co hoat tinh phong xa co th c c inh va lam kh trc khi phong xa t ghi. - Ngm gel cung vi tm kinh trong acetic acid 7% trong 5 phut. Ly gel khoi thuc ham mau mt cach cn thn bng cach cm nhe tm kinh ra khoi cht long. - Ra nhanh gel trong nc kh ion. Dung giy thm Kimwipe thm kh b mt gel (khng dung giy thm thng). Boc gel va tm kinh trong Saran wrap, thit k phong xa t ghi nh trn. Mt cach khac la lam kh gel trn giy Whatman 3MM o bng cach dung may lam kh gel (sy 80 C trong iu kin chn khng t 1-2 gi). Lam kh gel cn thit chi khi gel cha DNA c anh du ng vi phat xa yu nh 35 32 S hoc mt lng nho P (cn phai u phim 24 gi hoc lu hn) (Hnh 2.8).

1.3. Phn lp cac oan DNA t polyacrylamide gel

Phng phap tt nht phn lp DNA t polyacrylamide gel la ky thut p v ngm (crush and soak) cua Maxam v Gilbert (1977). DNA thu c co tinh sach cao va khng cha cac cht nhim bn c ch enzyme. Mc du phng phap nay phai tin hanh qua nhiu bc va khng hiu qua (it hn 30% san lng cac oan DNA >3 kb), nhng no co th c dung phn lp ca hai loai DNA si i va si n t cac polyacrylamide gel khng bin tinh va bin tnh, tng ng. Phng thc sau y c cai tin t ky thut cua Maxam va Gilbert (1977): - Chay in di polyacrylamide gel. Xac inh vi tri cua DNA quan tm bng phong xa t ghi hoc bng EtBr quan sat di anh sang UV. - Dung dao ct manh gel cha bng DNA quan tm. Khng loai bo Saran wrap khi gel trc khi ct, ct ca gel ln Saran wrap, sau o boc manh gel nho co cha DNA quan tm ra khoi Saran wrap. - Chuyn manh gel vao trong E-tube (eppendorf tube), dung tip cua micropipette ep manh gel theo thanh cua tube. - Tinh toan th tich thich hp cua manh gel va b sung 1-2 th tich cua m chit (0,5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA pH 8 va 0,1% SDS) vao E tube. - ong tube va u 37oC kem theo lc vong nhe. oan DNA nho (<500 bp) c dung ly trong khoang 3-4 gi, oan ln hn mt khoang 12-16 gi.

- Ly tm 12.000 g trong 1 phut 4 oC. Chuyn th ni vao E-tube sach.

- B sung thm 0,5 th tich m chit vao E-tube cu co manh polyacrylamide gel, vortex nhanh va ly tm. Trn hai th ni lai vi nhau. - B sung hai th tich ethanol 4oC, bao quan dung dich trn a tuyt 30 phut. Thu o hi DNA bng cach ly tm 12.000 g trong 10 phut 4 C. - Ha tan tr lai DNA trong 200 L m TE (pH 7,6) va b sung 25 L cua 3 M sodium acetate (pH 5,2) va kt tua DNA vi hai th tich ethanol nh bc trn. - Ra cn thn tiu th vi ethanol 70% va ha tan tr lai DNA trong m TE (pH 7,6) ti th tich cui cung la 10 L. - Kim tra s lng va cht lng cua oan DNA bng in di polyacrylamide gel. Ly mt th tich nho ti thiu (c c lng khoang 50 ng DNA) trn vi 10 L TE (pH 7,6), b sung thm 2 L gel-loading dye. Nap mu va chay in di polyacrylamide gel nng thich hp bng cach dung cac DNA marker chun a bit s lng. Cac oan DNA phn lp se cung dich chuyn vi DNA marker trong qua trinh in di.

2. in di protein 2.1. in di SDS-PAGE 2.1.1. Phng thc in di trn polyacrylamide gel vi s c mt ca SDS (sodium dodecyl sulfate) l k thut dng trong ha sinh, di truyn v sinh hc phn t phn tch cc protein theo tnh linh ng in di ca chng (ph thuc vo chiu di ca chui polypeptide hoc khi lng phn t, cu hnh (xon) ca protein, cc bin i hu dch m v cc nhn t khc). in SDS cho php phn ly cc phn t protein c khi lng khc nhau. SDS c in tch m rt ln v c kh nng lin kt vi mch peptide bin tnh cc cu trc bc hai v bc ba (khng lin kt bng cu disulfide) ca protein bng cch bc xung quanh khung polypeptide. Nh vy, s lng SDS tng tc vi protein t l vi khi lng/kch thc phn t protein v in tch ca SDS bm vo c th lm bt c phn t protein no cng chuyn ng trong in trng t cc m (-) sang cc dng (+). Do , bng phng php in di, c th phn tch ring bit cc phn t protein c khi lng phn t khc nhau. Khng c SDS, cc protein khc nhau c khi lng phn t tng t s dch chuyn khc nhau do s khc nhau v cun xon, bi v nhng khc nhau trong cc kiu cun xon s lm cho mt vi phn t protein thch hp tt hn vi khun gel so vi nhng phn t protein khc. B sung SDS gip gii quyt vn ny, v n lm cho cc phn t protein tr thnh mch thng sao cho chng c th phn tch hon ton theo khi lng phn t (cu trc s cp, hoc s lng (v kch thc) ca cc amino acid). SDS lin kt vi protein theo t l khong 1,4 g SDS trn 1,0 g protein (mc d t l lin kt c th thay i t 1,1-2,2 g/SDS/g protein) to ra mt t l khi

lng/in tch thng nht cho mi phn t protein s dch chuyn qua gel ch lin quan n kch thc ca protein. Mt loi thuc nhum vt c th c b sung vo dung dch protein cho php theo di tin ca dch chuyn protein qua gel trong sut qu trnh in di.

Hnh 2.9. in di protein bng k thut SDS-PAGE v nhum bng Coomassie blue. WM: Chun khi lng phn t ca protein. Cc ng 1, 2, 3, 4, 5, 6, 7 v 8: Mu protein.

2.1.2. Kh SDS-PAGE Bn cnh vic b sung SDS, protein c th c un nng nhanh gn n si vi s c mt ca mt tc nhn kh, v d nh dithiothreitol (DTT) hoc 2mercaptoethanol (BME), bin tnh thm protein bng cch kh cc lin kt disulfide, v th khc phc c mt vi dng cun xon bc ba ca protein, v b gy cu trc bc bn ca protein (cc tiu n v oligomer). Phng thc ny c xem nh l kh SDS-PAGE, v c s dng ph bin nht. Trng hp khng kh SDS-PAGE (khng un si v khng c tc nhn kh) c th c dng khi cu trc nguyn th l quan trng trong phn tch v sau (chng hn nh hot tnh enzyme, c trnh by bng vic s dng zymogram). in di polyacrylamide gel lin tc nguyn th pha ch nh lng (quantitative preparative native continuous polyacrylamide gel electrophoresis, QPNC-PAGE) l mt phng php mi phn tch cc metalloprotein nguyn th trong cc khun sinh hc phc tp.

2.1.3. in di v nhum Cc protein bin tnh sau c np vo mt u ca khun polyacrylamide gel t trong m thch hp. Dng in c s dng t u ny n u kia ca gel, s lm cho cc protein tch in m dch chuyn qua gel. Ty thuc vo kch thc ca chng, mi protein s dch chuyn vi cc tc khc nhau qua khun gel: protein

ngn d dng i qua cc l ca gel, trong khi protein ln hn s gp kh khn hn. Sau mt vi gi (ty thuc in p s dng trn gel, nu in p cao protein chy nhanh hn nhng s cho phn gii km hn), cc protein s c s dch chuyn khc nhau da trn kch thc ca chng, cc protein nh hn s i nhanh hn xung pha di ca gel, trong khi cc phn t ln vn cn v tr gn vi im xut pht. V th, protein c th c phn tch theo kch thc (v v th, theo khi lng phn t). Sau in di, gel c nhum (ph bin nht l Coomassie Brilliant Blue hoc thuc nhum bc), cho php quan st cc protein c phn tch, hoc nhng tin trnh xa hn (v d: Western blot, xem chng 7). Sau khi nhum, cc protein khc nhau s xut hin cc bng phn bit trong gel. Qu trnh in di thng c chy km vi protein marker ca cc khi lng phn t bit mt ng ring bit trong gel, canh chnh gel v xc nh khi lng ca protein cha bit bng cch so snh vi khong cch dch chuyn lin quan vi marker. in di polyacrylamide gel thng l chn la u tin khi th nghim s tinh sch protein do tnh ng tin cy v d dng ca n. S c mt ca SDS v bc gy bin tnh lm cho cc protein c phn tch n c da trn kch thc. Cc tc nhn bn cng c th cng dch chuyn v xut hin mt bng khng mong mun tng t protein. S ng dch chuyn ny cng c th lm cho mt protein s chy mt v tr khc hoc khng th xm nhp vo gel. y l iu quan trng nhum gel hon ton bao gm phn stacking. Coomassie blue c i lc lin kt yu vi glycoprotein v cc protein dng si, lm cn tr cht lng in di.

2.1.4. H thng m Hu ht in di phn tch protein c thc hin vi mt h thng m khng lin tc tng cng mt cch ngha hnh dng ca bng trong gel. Trong sut qu trnh in di h thng gel khng lin tc, mt gradient ion c to thnh trong giai on sm ca in di lm cho tt c protein tp trung trong mt bng dng n. Nhiu ngi s dng lin tc m Tris-glycine hoc Laemmli tp trung v phn gii pH 8,3-9,0. Cc pH ny khi ng s to thnh cu ni disulfide gia cc gc cysteine trong protein, c bit khi chng hin din nng cao do pKa ca cysteine nm trong phm vi t 8-9 v bi v tc nhn kh hin din trong m np (loading buffer) khng ng dch chuyn vi cc protein. Nhng tin b gn y trong cng ngh m lm nh bt vn ny bng cch ha tan protein pH tt di pKa ca cysteine (v d: bis-Tris, pH 6,5) v bao gm cc tc nhn kh (v d: sodium bisulfite) l yu t di chuyn vo gel pha trc cc protein duy tr mt mi trng kh. Mt li ch na ca m c s dng vi cc pH thp l acrylamide gel n nh hn v th gel c th c bo qun trong mt thi gian di trc khi s dng.

2.2. in di 2D-PAGE Ngoi in di SDS, c th in di cc protein ty theo im ng in (isoelectric point, IEP) ca chng. Phng php ny c gi l in di tp trung ng in (isoelectric focusing, IEF). Trong dung dch m c pH bin thin lin tc (gradient pH), cc protein s phn ly n v tr tng thch vi im ng in ca mnh. Mt hn hp protein phc tp c np vo gia ca mt tri, pH l trung tnh v mt in p c s dng trn khun gel. Cc protein sau dch chuyn thng qua gel cho n khi chng hng ti im ng in ca mnh, l im m in tch ca chng tng t nh pH vng chung quanh. Gel sau c ngm trong dung dch bin tnh ( lm cho cc protein khng cun xon) cha cht ty l SDS. Phn t ny tch in m rt mnh v lin kt vi tt c protein, lm cho tt c chng u mang in m. Mt in p sau c s dng trn khun gel t u ny n u kia tt c protein s chuyn ng hng ti anode, nhng nhng protein nh hn s chuyn ng qua gel nhanh hn, v th cc protein c phn tch theo kch thc ca chng. K thut in di 2 chiu (2D-PAGE, 2 dimension polyacryamide gel electrophoesis) c dng phn tch hn hp protein, v c bit hu ch cho vic so snh cc mu lin quan nh mu m bnh thng v mu m t bin. Comparative 2D-PAGE cng c th c dng tm kim cc protein m s biu hin ca chng rt tng ng di cng mt tp hp cc iu kin (nhng yu t ny c cc chc nng lin quan) v nhn dng cc protein c sn xut trong phn ng vi liu php thuc.

Hnh 2.10. in di protein hai chiu

C khong 10.000 protein khc nhau trong t bo, tp trung trong hn su loi quan trng. K thut 2D-PAGE khng nhy pht hin cc protein him v nhiu protein s khng c phn gii. V th, cn phi phn ct mu trong cc phn khc nhau lm gim s phc tp ca hn hp protein trc khi thc hin 2D-PAGE. Hai phng php in di theo khi lng phn t v im ng in c th kt hp vi nhau to nn k thut in di 2 chiu. in di protein trn polyacrylamide gel cho php phn on, xc nh khi lng phn t v phn lp protein. Ngoi ra, khi lng protein cn c xc nh chnh xc bng phng php sc k khi ph. Ti liu tham kho/c thm
1. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA and Struhl K. 2002. Short Protocol in Molecular Biology. Vol 1 and 2. 5th ed. John Wiley & Sons, Inc. USA. 2. Maniatis T, Fritsch EF and Sambrook J. 2000. Molecular Cloning-A Laboratory Manual. Cold Spring Habor Laboratory Press, USA. 3. Rapley R and Walker JM. 1998. Molecular Biomethods Handbook. Humana Press Inc. New Jersey, USA. 4. Rickwood D and Hames BD. 1984. Gel Electrophoresis of Nucleic Acids. IRL Press Ltd. Oxford, UK. 5. Surzycki S. 2000. Basic Techniques in Molecular Biology. Springer-Verlag, Berlin, Heidelberg, Germany.

6. Westermeier R. 2005. Electrophoresis in Practice. 4th ed. Wiley-VCH Verlag GmbH & Co. KgaA. Weinheim, Germany.

Agar: mt galactan phc hp tch chit t mt s loi to nh Gelidium v Gracilaria spp., c s dng rng ri dng gel lm gi th rn hoc bn rn cho mi trng nui cy vi sinh vt v thc vt.
1

Chng 3

Khuch ai in vitro DNA bng phan ng chui polymerase (PCR)

PCR (polymerase chain reaction) l mt k thut c s dng ph bin trong cng ngh sinh hc hin i v ng gp rt ln cho nhng tin b v sinh hc phn t, nh du mt bc tin v cung quan trng tng ng vi vic khm ph ra cc enzyme hn ch (xem chng 1) v k thut Southern blot (xem chng 5). PCR da trn c s phan ng ko di primer nh enzyme Taq polymerase khuch ai in vitro cac nucleic acid c hiu trong thit b iu nhit tun hon (thermocycler) cn gi l my PCR (Hnh 3.1). PCR cho phep khuch ai theo ham mu ln n hang triu ln cac oan DNA co chiu dai t 200-3.000 bp. oan DNA c khuch ai (DNA ich) c nhn dng nh cp primer c hiu (oligonucleotide) thng co chiu dai khoang 20 nucleotide.

Hnh 3.1. Thit b iu nhit tun hon

I. Nguyn tc ca PCR

Taq polymerase (xem chng 1) l mt loi enzyme DNA polymerase chu nhit (c vi khun chu nhit cao Thermus aquaticus) c dng tng hp cc on DNA mi trong mi trng co bn loi deoxyribonucleotide (dATP, dCTP, dGTP v dTTP) v hai primer, trn c s khun mu ca mt on DNA nht nh a bit hoc cha bit trinh t. Cc on DNA mi hnh thnh li c s dng lm khun mu. Sau nhiu chu ky, s lng on DNA ni trn c nhn ln gp nhiu ln, nh vy c th s lng tch ra, phn tch trnh t hoc to dng... Primer bn trai tac ng trn si DNA 3-5 c goi la primer thun (forward primer, ky hiu la F). Primer bn phai tac ng trn si DNA 5-3 c goi la primer ngc (reverse primer, ky hiu la R). Nguyn tc ca PCR c trnh by trong hnh 3.2 v 3.3. Theo , t chu ky th hai Taq DNA polymerase (gi tt l Taq pol) bt u to ra cc on DNA c chiu di xc nh. Cc primer thng l mt oligonucleotide tng hp (synthetic oligonucleotide) c khong 10-20 nucleotide hoc hn. Nu bit trnh t ca on gen cn khuch i th c th tng hp nhn to cc primer tng ng thc hin PCR v tch chng ra bng k thut in di. PCR thng tin hnh khoang 25-35 chu ky, qua t 10 -6 g DNA ban u c th khuch i (amplification) ln ti trn 1 g (khong 2 kb). Mi chu ky PCR bao gm ba giai on c nhit khc nhau: - Gy bin tnh (denaturation) 90-95oC Trong giai on bin tnh, phn t DNA khun mu dng xon kp c tch thnh hai si n (single strands). Tt c cc phn ng enzyme trong giai on ny u b dng li (v d: phn ng tng hp DNA t chu k trc ). - Gn mi (annealing) 40-65oC Trong giai on ny cc primer gn vo cc v tr c trnh t tng ng DNA khun mu. Cc primer b lc nh chung quanh do chuyn ng Brown v th cc lin kt ion c to thnh v b t gy lin tc gia primer si n v DNA khun mu si n. Cc lin kt ion n nh hn tao thanh mt on nh (cc primer lp rap chnh xc) v trn cc on nho DNA si i (khun mu v primer) Taq pol c th bt u qu trnh sao chp khun mu. giai on ny phm vi nhit c s dng c th rt rng ty thuc vo trnh t nucleotide ca primer, thng thng khong 55oC, nhng c khi ch 35oC hoc i lc ln n 68oC. - Ko di phn t (extension) nhit 70-72oC. y l khong nhit ti thch cho Taq pol tin hnh tng hp DNA bng cch b sung cc dNTP bt u t cc v tr c primer theo chiu 53. Cc primer c mt vi base gn vo khun mu c mi lin kt ion mnh hn lc ph v cc lin kt ny se khng bi t gay. Cc primer cc v tr khng bt cp chnh xc li b ri ra (do nhit cao) khi khun mu a khng tng hp c DNA.

Hnh 3.2. S phn ng chui polymerase

Hnh 3.3. Cc chu k ca k thut PCR

Co ba ky thut PCR thng dng: PCR chun, PCR mo neo va PCR ao ngc. Trong PCR chun (standard PCR), DNA khun mu c m xon kep, sau o hai primer tac ng hai u si n, ri DNA mi c tng hp nh Taq pol vi 4 loi deoxyribonucleotide (Hnh 3.4).

Hnh 3.4. S k thut PCR chun

PCR mo neo (anchored PCR), k thut ny yu cu ch cn bit trnh t nucleotide mt u ca khun mu v gn mt ui ng trng hp nhn to (v d: dA) vao u kia (cha bit trnh t). Sau on DNA c khuch ai nhiu ln nh mt primer co trinh t a bit trc va mt primer th hai co oligo(dT) (Hnh 3.5). PCR ao ngc (inverse PCR), c dung khuch ai cc oan phn t k cn vi on c trinh t DNA a bit trc, phn t DNA nay c ct hn ch va ni lai nh enzyme DNA ligase tao thanh mt vong tron co tinh cht monomer, sau o on DNA c khuch ai nh hai primer tng ng vi u cua trinh t bit trc (Hinh 3.6).

II. Quy trnh PCR 1. Thnh phn phn ng - Phn t DNA c cha on DNA cn khuch i - 2 primer (F v R) - Taq pol - 4 loi dNTP - m v cc mui khong

Hnh 3.5. S k thut PCR m neo

2. Thc hin phn ng Di y la vi du minh ha cho mt phan ng khuch ai:

2.1. Chun b dung dich master mix v cho vo eppendorf tube (E-tube) loi 0,5 mL, trn u cc thnh phn sau: m 10 PCR 4,5 L Hn hp 4 dNTP (2,5 mM mi loai) 4 L Primer-F (10 pmol/L) 2,5 L Primer-R (10 pmol/L) 2,5 L Thm H2O ti 40 L 2.2. Chun b dung dich pha loang cua Taq pol: m 10 2 L Taq pol (5 units/L) 1 L Thm H2O ti 20 L

Hinh 3.6. S ky thut PCR ao ngc

2.3. B sung 5 L DNA khun mu (100 ng) vo 40 L dung dch ca master mix tube. 2.4. B sung 2 L/1 tube dung dich pha loang cua Taq pol. 2.5. t E-tube ng dung dch phn ng vo heating block ca my PCR thc hin ch nhit theo chu k nh sau: - Start program - 95oC/5 pht - Thc hin 30 chu ky: 95oC/30 giy, 50oC/30 giy va 72oC/1 pht - 72oC/10 pht - Gi san phm PCR 4oC - Chy in di kim tra kt qa PCR (Hinh 3.7) - Nu cha chay in di thi bao quan san phm PCR -20oC
Ch y - Cc thnh phn v iu kin ca phan ng nh: di ca primer, ch nhit trong mt chu ky, s chu ky i vi mi i tng c th thay i t nhiu bng thc nghim. Cc thng s trn chi c y nghia tham kho.

- Nu lm nhiu mu, c th trn chung cc thnh phn khc tr DNA v Taq pol, sau chia ra tng tube ri cho DNA v Taq pol vao sau cng. - Thao tc trong t cy v trng (hoc khng).

Hnh 3.7. in di cc sn phm PCR. SM: Chun kch thc DNA. Cac ng s 1, 2, 3, 4 va 5: Cc san phm PCR khc nhau.

III. Ti u hoa cac iu kin cho PCR 1. Trinh t cua primer i vi genome cua eukaryote ngi ta thng dung cac primer dai khoang 18 nucleotide tr ln. Tuy nhin, cung tuy tng trng hp, co nhng primer cua cac ch th 1 2 phn t ngu nhin nh RAPD rt ngn (10-16 mer) hoc STS cn primer di hn (20 24 mer). Noi chung, genomic DNA khng hoan toan la mt trinh t ngu nhin ma no con mang c tinh cua cac ho gen, nhng nhn t co tinh lp lai (s lp oan n gian hay phc tap). Tuy theo loai sinh vt va tuy theo cach th hin cua trinh t DNA khun mu, ngi ta se thit k trinh t cua primer va i chiu cn thn no vi s liu lu tr trc y nhm loai tr cac sai sot v ky thut. Gn y, ngi ta thng s dung phn mm trong computer thit k cac primer thich hp cho tng muc tiu nghin cu va co th giup loai bo cac cp primer c thit k khng ti u. Khi thit k primer cn chu y mt s im nh sau: C gng chon trinh t primer co khoang 50% GC. Tranh G va C u 3 cua primer vi no co th lam tng c hi tao ra hin tng primer-dimers (hai primer bt cp vi nhau). Tranh chon cac vung co trinh t tng ng han ch cac primer t gn vi nhau. Tinh toan nhit nng chy (Tm) o o cua primer vi tng s 4 C cho GC va 2 C cho AT, sau o tr i 5 oC t gia tri nay va y chinh la nhit u (Ta) cua primer. Noi chung, nhit u co gia tri thp hn nhit nng chy ca primer. S khac nhau t 4-6 oC gia Tm primer va Ta dng nh khng anh hng n hiu sut cua PCR.

2. Nhit cua qua trinh u Nhit u thich hp l nhit sao cho o 1/2 s primer se gn vi DNA khun mu. Cng thc di y ng dung trong trng hp primer co khoang 20 oligonucleotide: Ta = 4 (G + C) + 2 (A + T) 5oC (1) Tuy nhin, cng thc nay chi co tinh tng i, bng kinh nghim nghin cu chung ta mi co th co s liu ung v nhit cho qua trinh u. S base cua primer cang it thi nhit nay cang thp va ngc lai. 3. Magnesium Nng cua Mg2+ (c cung cp di dang MgCl2) co th anh hng n nhit bin tinh DNA khun mu, qua trinh u cua primer, tinh c hiu cua san phm PCR, hoat tinh cua Taq pol va chinh xac cua kt qua. Nng thich hp cua Mg 2+ la t 0,5 2+ 2,5 mM ng vi nng dNTP tng s a cho. Nng Mg cao se lam cho phn t DNA si i n inh hn va ngn nga c s bin cht hoan toan (m xon giai phong si n cua san phm PCR trong mi chu ky) lam cho kt qua PCR ngheo i. Mt khac, no con lam cho hin tng bt cp gia (tai nhng vi tri khng tng ng) n inh hn dn n xut hin nhng san phm PCR khng c hiu vi s lng kha ln. 2+ Ngc lai, nu nng Mg qua thp se anh hng xu n qua trinh tng hp DNA (Mg2+ ong vai tro la mt co-factor cua Taq pol). Do o, cn phai xac inh nng ti u 2+ cua Mg nhm am bao hiu sut khuch ai va tinh c hiu cua san phm PCR.

4. Cac deoxyribonucleotide triphosphate (dNTPs) Dung dich stock ca cc dNTP phai co pH 7, nng thng dung la 10 mM (2,5 o mM mi loai) c bao quan -20 C. Ham lng cua cc dNTP trong khoang 20-200 M cho kt qua n inh, chinh xac va c hiu. Bn loai dNTP c s dung cn co nng tng ng nhau giam thiu ti a hin tng sai bit do kt hp sai (misincorporation) ma di truyn nao o.

5. Enzyme Taq pol Nng Taq pol thich hp la t 1-2,5 unit cho 100 L dung dich phan ng. Thng thng co th s dung 0,5 unit/25 L. Nu nng Taq pol qua cao, co th xut hin cac san phm PCR khng c hiu va lam sai lch kt qua. Nu nng Taq pol qua thp, se khng u lng enzyme xuc tac tao ra san phm PCR mong mun.

6. m n inh hoat ng cua Taq pol Nhng cht n inh Taq pol c s dung la gelatin va Triston X-100 trong m bao quan enzyme. Nng thng dung la 0,01% gelatin va 0,1% (v/v) Triston X-100.

7. Nng cac primer Trong hu ht cac ng dung cua PCR, hai primer F va R u phai co nng bng nhau. Nng cui cung cua mi primer la 0,1 M, tng ng vi 2,5 pmol (16,25 ng cua 20-mer) trong 25 L dung dich phan ng. S dung nng primer cao khng cn thit va thng tao ra bt li, vi qua tha primer se co hin tng primer-dimers va hin tng gn primer nhm vi tri trn khun mu.

8. Nng DNA khun mu PCR bao gm hai giai oan: giai oan sang loc va giai oan khuch ai. Nu cac cht trong thanh phn phan ng (bao gm primer) co nng cao trong khi DNA khun mu lai co nng thp thi giai oan sang loc cua PCR se tr nn kho khn hn, vi tn sut primer va DNA khun mu gp nhau giam ro rt, trong khi s tip xuc gia primer va primer lai tng ln gy ra hin tng primer-dimers trong giai oan khuch ai. Thng thng, nng DNA khun mu c s dng trong khong t 10-100 ng/25 L dung dch phn ng.

9. Ty l gia primer va DNA khun mu Mt trong nhng yu t quan trong nht cua PCR la ty l ti u gia primer va DNA khun mu. Nu ty l nay qua cao thi hin tng primer-dimers se xut hin, ging nh trng hp mu DNA qua loang. Nu ty l nay qua thp kt qua san phm PCR se khng nhiu. Trong hu ht cac trng hp ap dung PCR, ngi ta u dung nng primer khng qua 0,5 M (12,5 pmol/25 L) va tinh toan nng DNA khun mu tranh hin tng primer-dimers.

10. Khi ng nng PCR Khi ng nong PCR (host-start PCR) la phng phap san xut cac san phm PCR sach hn. DNA khun mu va primer c trn vi nhau va gi nhit trn ngng cua lin kt khng c hiu gia primer va khun mu. Tt ca cac thanh phn

cua PCR c b sung trc ngoai tr mt cht then cht (thng la Taq pol). Chi trc khi i vao chu ky khuch ai, thanh phn cha co mi c b sung cho phep phan ng xay ra nhit cao hn. Do khng co hin tng lai khng c hiu cua primer vi khun mu, nn cac bng DNA c khuch ai tr nn sang hn, cac oan primer khng co c hi gn vi nhau. Ky thut nay c tin hanh bng cach lam lanh cac tube trn tuyt (ice bath) trong khi b sung hn hp thanh phn cua PCR. Sau o, t tube trong may PCR a c lam nong ngay trc khi b sung thanh phn cui cung. Di y la vi du minh ha cho khi ng nng PCR.

10.1. Chun b dung dich master mix v cho vo E-tube loi 0,5 mL v trn u cc thnh phn sau: m 10 PCR 4,5 L Hn hp 4 dNTP (2,5 mM mi loai) 4 L Primer-F (10 pmol/L) 2,5 L Primer-R (10 pmol/L) 2,5 L Thm H2O ti 40 L 10.2. Chun b dung dich pha loang cua Taq pol: m 10 2 L Taq pol (5 units/L) 1 L Thm H2O ti 20 L 10.3. B sung 5 L DNA khun mu (100 ng) vo 40 L dung dch ca master mix tube.

10.4. t E-tube ng dung dch phn ng vo heat block ca my PCR thc hin ch nhit theo chu k nh sau: - Start program - Hot start: Khong 80oC/10 giy pause - B sung 2 L/1 tube dung dich pha loang cua Taq pol. - Restart - 94oC/5 pht - Thc hin 30 chu ky: 94oC/30 giy, 54oC/30 pht va 72oC/1 pht. o - 72 C/10 pht - Gi san phm PCR 4oC - Chy in di kim tra kt qa PCR

- Nu cha chay in di thi bao quan san phm PCR -20oC

IV. Thit k primer Vic chon la mt primer c mt ngha quan trng khi mun ng dung PCR co hiu qua va chinh xac cao. Vi th, chung ta phai co mt thit k chinh xac v oligonucleotide primer. Trinh t cua primer c chon xac inh kich thc v vi tri cua san phm PCR, cung nh Tm ca vung c khuch ai. Primer c thit k ung co th giup chung ta tranh tao ra nhng san phm PCR khng c hiu (non-specific). Muc ich cua vic thit k primer la co c mt s cn bng gia hai kt qua: s c hiu va hiu sut khuch ai. S c hiu c xem xet trn c s xut hin bao nhiu ln hin tng gn primer nhm (mis-priming) trn tng s ln gn primer (priming) tc la s lai gia primer va khun mu tai vi tri ich cua no. Hiu sut la s tip cn vi ly thuyt v kt qua san phm, trong mi chu ky PCR, mt cp primer co th khuch ai ra mt san phm. Vi mt trinh t DNA a c cung cp ngi ta co th phn tich primer trn computer co mt kt qua cn bng gia hai muc tiu noi trn.

1. Chiu dai primer Tinh c hiu cua san phm PCR thng phu thuc vao chiu dai cua primer va nhit u. Cac oligonucleotide co kich thc khoang 18-24 base co xu hng tr thanh nhng trinh t rt c hiu nu nhit u cua PCR c thit k sai khac chi vai so vi Tm cua primer (nhit ly thuyt). Primer c chiu di cng ln th kha nng gn ca primer cng nh. Trong khi khuch i, mt s c no ca qu trnh cng s lm gim kt qu PCR. ti u ha PCR, ngi ta s dng primer c chiu di ti thiu sao cho m bo Tm khong 54oC hoc hn cht t, iu ny s cung cp nhng c hi tt nht duy tr tinh c trng ca sn phm PCR v hiu sut phn ng cao. Nhng oligonucleotide ngn (15 base hoc ngn hn) c s dung hn ch trong nhiu quy trnh PCR. Chiu di primer ti thiu cho hu ht cc ng dng PCR l khoang 18 nucleotide va ngi ta thng thit k cac primer c di 18-24 mer. Primer cng ngn th qu trnh gn gia primer v khun mu DNA cng nhanh, to thnh si i n nh trong tng hp DNA. Thng thng, cac primer c 28-35 mer cn cho vic khuch i cc trnh t c mc d hp cao. i vi nhng primer ngn hn 20 mer, c th s dng cng thc tnh Tm lin h vi cc base: Tm = 4 (G + C) + 2 (A + T). Trong khi primer di hn cng thc ny ch c gi tr gn ng.

2. Nucleotide cui cung cua primer

V tr u 3 ca primer nn c xc nh cn thn, v n c tnh cht quyt nh cho s thnh cng ca PCR. Khi chng ta xc nh c mt amino acid, hai base u tin trong codon ca n hoc ba base trong trng hp n c m ha bng codon n (methionin v tryptophan) th hai hoc ba base u tin ny c dng nh u 3. Nhng cp base hon chnh gia nhng u 3 ca primer v khun mu cho php chng ta c c kt qu mong mun, v gim thiu ti a hin tng bt cp sai (mismatch) trong khong 5-6 nucleotide ti u 3 ca primer. Thng thng, vic thm vo mt trnh t khng lin quan ti u 5 ca primer vn khng lm thay i qu trnh gn mi. Nhim v ca u 3 trong primer l kim sot hin tng gn primer nhm va ngn can hin tng tng ng trong cng mt cp primer. Nu khng thn trng trong vic xc nh u 3 thi hin tng primer-dimers s xy ra, va vi tnh cht b sung cho nhau sn phm PCR lc by gi se l mt s khuch i ca chnh primer ch khng phi cua DNA khun mu m ta mong mun. Trong trng hp c nhiu cp primer a vo trong cng mt phn ng (multiplex PCR), chng ta cn phi kim tra gp i hin tng b sung cho nhau ca tt c primer.

3. Ham lng GC va nhit nong chay Tm Primer ca PCR phi duy tr c hm lng GC n mc c th c. Nhng oligonucleotide c 20 base vi 50% GC thng c gi tr Tm trong khong 56-62oC se to iu kin qu trnh t kt qu tt. Hm lng GC v Tm phi khp vi mt cp primer c thit k. Nu gi tr Tm cng ln thi c hi cho hin tng gn primer nhm cng cao. Do , khi thit k primer cn phi lu c bit n hm lng GC.

V. K thut RT-PCR Phn ng RT-PCR l phn ng khuch i mt on khun mu RNA theo nguyn l ca PCR, bao gm hai giai on: Giai on th nht. Phin m ngc khun mu RNA thnh si DNA th nht, sau dng si ny lm khun mu tng hp si DNA th hai (tng t nh giai on th nht v th hai ca qu trnh tng hp cDNA-xem chng 6). Giai on th hai. Dng DNA si i lm khun mu thc hin phn ng PCR nh trnh by trn. RNA c cu to nh bn loi nucleotide (adenine, guanine, cytosine v uracil) lin kt vi nhau to thnh mt chui n. Khi chui nucleotide ny c bin i thnh DNA th uracil (U) c thay th bng thymine (T). Phn ng bin i RNA h gen thnh si DNA th nht phi nh n mt enzyme phin m ngc (reverse

transcriptase) do vy giai on ny c gi l phin m ngc (RT), sau qu trnh tng hp si DNA th hai li nh mt enzyme khc l DNA polymerase I (thng dng on Klenow). Khi c DNA si i t khun mu RNA th phn ng tip theo s l khuch i gen nh k thut PCR c thc hin vi ba mc nhit ph hp mi chu k. Ton b phn ng khuch i mt on DNA t khun mu RNA tri qua hai giai on ni trn c gi l RT-PCR. Do trong t nhin mt s virus c h gen l RNA (retrovirus) nn mun khuch i mt on gen ca n th ngi ta phi dng k thut RT-PCR. Mt s nghin cu khc v mRNA cng cn n RT-PCR bin i si RNA thnh DNA cho qu trnh to dng phn tch trnh t gen (gene sequencing) hay ti t hp (recombination) trong nghin cu biu hin gen.

VI. Real-time PCR 1. Gii thiu chung Trong PCR truyn thng, sn phm khuch i c pht hin nh s phn tch u cui (end-point) bng cch chy in di DNA trn agarose gel sau khi phn ng kt thc. Ngc li, phng php real-time PCR cho php pht hin v nh lng sn phm khuch i khi tin trnh phn ng ang din ra da trn c s phn ng hunh quang, trong s tng ln v s lng DNA tng ng vi s tng ln ca tn hiu hunh quang. Trong real-time PCR, ngi ta thng s dng cc loi thuc nhum lin kt DNA si i (SYBR Green) pht hunh quang, v cc probe hoc primer c hiu chui (trnh t) c nh du hunh quang (TaqMan PCR). Thit b n nhit chu k (my PCR) c bit c gn vi mt module pht hin tn hiu hunh quang theo di tin trnh phn ng khi s khuch i xy ra. Tn hiu hunh quang o c phn nh s lng sn phm c khuch i trong mi chu k. Real-time PCR c u im chnh so vi PCR truyn thng l cho php chng ta nh lng s copy khi u ca khun mu vi chnh xc v nhy cao trn mt phm vi ng hc ln. Cc kt qu ca real-time PCR c th l cht lng (s c mt hoc khng ca trnh t ch) hoc nh lng (s bn copy ca DNA). Real-time PCR nh lng cn c bit nh l qPCR. S liu ca real-time PCR c th nh gi m khng cn in di gel, thi gian th nghim c rt ngn v s lng nguyn liu a vo qu trnh c tng ln. Cui cng, do cc phn ng c chy v s liu c nh gi trong mt h thng tube ng kn, nn cc c hi nhim bn c gim thiu v s cn thit ca cc thao tc hu khuch i c loi b.

2. Phn tch tng qut mt phn ng real-time PCR

 hiu c real-time PCR nh th no, chng ta bt u bng mt ng cong khuch i mu (Hnh 3.9). Trong hnh ny, s chu k PCR c trnh by trn trc x, v tn hiu hunh quang t phn ng khuch i, t l vi s lng sn phm c khuch i trong tube, c trnh by trc y. ng cong khuch i c 2 pha, mt pha hm m (exponential phase) c tip theo bi mt pha n nh (plateau phase). Trong sut pha hm m, s lng sn phm PCR xp x gp i trong mi chu k. Tuy nhin, khi phn ng din ra thnh phn phn ng s b tiu hao, cui cng lm cho mt hoc nhiu thnh phn b hn ch. im ny phn ng chm li v i vo pha n nh (cc chu k 28-40, Hnh 3.9).

Hnh 3.9. ng cong khuch i. Tn hiu hunh quang c trnh by tr ng nn (baseline).

u tin, tn hiu hunh quang duy tr mc nn (background), v vic tng tn hiu hunh quang l khng th pht hin c (cc chu k 1-18, Hnh 3.9) cho d sn phm tch ly theo hm m. Sau , sn phm khuch i tch ly t ti mt hiu sut cho php pht hin c tn hiu hunh quang. S chu k t c hiu sut ny gi l chu k ngng (threshold cycle, CT). Do gi tr CT c o trong pha hm m khi cc tc nhn phn ng khng b hn ch, nn real-time qPCR c th c s dng tnh ton chnh xc v tin cy s lng khi u ca khun mu hin din trong phn ng. CT c xc nh ch yu bi s lng ca khun mu hin din lc bt u phn ng khuch i. Khi lng khun mu nhiu th ch cn mt vi chu k khuch i tch ly sn phm cho mt tn hiu hunh quang trn background. V vy, phn ng s c CT thp hn hoc sm. Ngc li, nu lng khun mu lc bt u

phn ng qu t, th s chu k khuch i cn nhiu hn tn hiu hunh quang tng trn background. V vy, phn ng s c CT cao hoc mun. Cc dng quan h ny l c s cho hng nh lng ca real-time PCR.

3. Mt s ng dng chnh ca real-time PCR - Nghin cu biu hin gen. Xc nh s hot ng ca gen v nh lng mc biu hin ca n thng qua RNA bng k thut RT-PCR. - Chn on phn t. Nghin cu mc nhim virus v vi khun chn on cc bnh vim nhim. - Xt nghim phn bit allele. L phng php xt nghim u cui c dng xc nh kiu gen ca mu vt (ng hp t: ch c allele 1 hoc ch c allele 2, d hp t: c c hai allele 1 v 2) v phn bit s a hnh nucleotide n (single nucleotide polymorhism, SNP). VII. Mt s ng dung cua PCR PCR c pham vi ng dung rt rng trong cng ngh sinh hc hin i. Theo nguyn tc trn, a c nhiu b sung va cai tin dung PCR cho nhiu muc ich khc nhau. y chi gii thiu mt s ng dung chnh: - Trong nghin cu genome + Nhn ban phn t bng PCR + PCR tai t hp + Ky thut footprinting DNase I + Sang loc th vin gt11 + Multiplex PCR... - Trong y hoc + Phat hin t bao T/virus gy bnh ung th mau + Phat hin virus vim gan B + Phat hin virus Dengue gy bnh st xut huyt + Phat hin vi khun lao...

1. S dung PCR tao nhanh cac gen tng hp S dung phng phap PCR hai giai oan (two-step PCR) tao nhanh cac gen tng hp. Phng phap ny c xy dng trn c s nhng nucleotide chng ln nhau (overlapping), do co th c s dung tao ra on DNA tng hp thng qua nhiu vong khuch ai PCR. Kha nng tao ra DNA tng hp co nhiu ng dung tuy thuc vao

thit k cua chung ta. Ngi ta co th thit k mt gen tng hp co cha codon c dung biu hin protein trong c th sinh vt. Phng phap nay c hoan tt nh s giai ma ngc chui amino acid cua protein mong mun. thay i cu truc cua codon, gen tng hp co th c thit k co nhng vi tri ct han ch thun li phuc vu cho thi nghim tao dong v sau. Nguyn tc cua PCR hai giai oan o la hai phan ng PCR xay ra lin nhau, phan ng th nht cua PCR phat sinh ra si khun mu tng ng vi gen tng hp va sau o no c khuch ai trong phan ng PCR ln th hai (Hnh 3.10). Trc khi bt u qua trinh nay, ngi ta phi thit k cu truc va xac inh trinh t nucleotide cua gen tng hp ma minh mong mun. Sau o, trinh t cc oligonucleotide (primers) theo chiu dai cua gen phai c thit k va tng hp. Thng thng, ngi ta tng hp cc oligonucleotide theo s chn vi chiu dai khoang 16-30 nucleotide.

2. Tao dong cDNA bng PCR Ky thut tao dong kinh in cDNA oi hoi nhiu cng sc v xy dng th vin bao quan bacteriophage hoc plasmid, no cn mt khi lng cng vic chn lc mt s lng ln cac phage hoc plasmid co tinh cht tai t hp. Co ba han ch trong phng phap nay: - Cn co mt lng c cht (it nht 1 g) mRNA tinh sach lam vt liu khi u xy dng th vin vi mc a dang y u cho nghin cu. - Kho khn thuc v ban cht bn trong cua nhng phan ng enzyme tip ni nhau lam cho vic tao dong cDNA co kt qua thp, va nhng dong gen bi t oan. - Vic sang loc mt th vin vi ky thut lai oi hoi nhiu thi gian. Ky thut PCR hin nay co th giup chung ta khc phuc nhng nhc im noi trn, lam cho vic tao dong cDNA tr nn d dang hn. S dung hai primer c hiu cua gen, mt cDNA co trinh t a c bit ro, ngi ta cho khuch ai trong PCR. Tuy nhin, cai kho la lam sao phn lp c nhng ban sao cua cDNA hon chnh (full-length) t mRNA, trn c s thng tin v trinh t rt han ch. Trinh t cha bit nay nm k mt oan nho cua trinh t a bit ro. Trinh t cha bit khng th khuch ai bng phng phap PCR thng thng. Do , phng phap PCR mo neo va phng phap PCR ao ngc a c phat trin trong thi gian gn y giai quyt vn nay.

Hnh 3.10. S minh ha mt phn ng to nhanh gen tng hp thng qua phng
php PCR hai giai on. Gen quan tm c khuch i trong phn ng PCR th nht vi cp oligonucleotide c hiu gen nhng c a vo vng chng ln nhau (primer A v primer B). Trong phn ng PCR th hai, cc primer m rng C v D gn vi vng chng ln nhau ca sn phm PCR ln th nht v b sung tt c cc nhn t iu ha cn thit cho s phin m v dch m. Sn phm PCR ln th nht c pha long 200 ln lm khun mu cho phn ng PCR th hai.

2.1. PCR mo neo Ky thut PCR mo neo co mt im chung la: Tao dong DNA i t mt oan trinh t a bit ri ti oan trinh t k cn cha bit nh s giup cua mt primer c hiu i vi gen tai mt u si n va mt primer tng hp tai mt u si n khac. Do chi co primer c hiu i vi gen mi co th neo trong PCR lam d dang hn cho vic thu thp mt lng ln san phm khuch ai khng c hiu so vi PCR chun. K thut ny khac vi PCR chun la chi cn mt primer c hiu cho phan ng. Trong ky thut PCR m neo, mt ui nhn tao cua cac gc dA c thm vao oan cui cua DNA khun mu. S khuch ai DNA xay ra khi co primer cua mt trinh t a bit trc va mt primer th hai co cac gc oligo(dT).

2.2. PCR ao ngc Phng phap PCR ao ngc co thun li ln hn nh khuch ai trinh t k cn cha bit bng hai primer c hiu cua gen. PCR ao ngc hoat ng theo nguyn tc tao dong si n mt trinh t DNA. Sau o, DNA nay c ct bng RE vi tri tng ng tao ra DNA ich, tip theo no hoat ng theo chu trinh co tinh cht monomer. DNA nay c khuch ai ln bng cac primer tng ng tai hai u si n cua DNA co trinh t a bit.

Hinh 3.11. ng dung PCR ao ngc. Hai primer c hiu c gn vo trnh t bit va sau o DNA c khuch i theo chiu mui tn.

Ban u la si 53cua mRNA cho vao phan ng phin ma ngc thanh si n cDNA 35, tip tuc tng hp si n cua cDNA ln th hai, cho ra si i cDNA (53va 35). Si nay co cha mt trinh t a bit ro. Ky thut tao dong lam cho cDNA thng bin thanh cDNA vong si i. Tip n, m vong nh RE ct trinh t c bit lam i, mt na u si thng bn nay, mt na bn kia. Keo thng si DNA va khuch ai trong PCR (Hinh 3.11).

3. ng dung trong di truyn PCR gip c lc cho vic lp ban gen (gene mapping) ca sinh vt. Phng php ny c tn l phn tch DNA a hnh c khuch i ngu nhin (RAPD). Trong phng php RAPD, PCR c thc hin vi cc primer chn ngu nhin. Kt qua l to ra mt s on DNA c chiu di khc nhau c trng cho mi loi, thm ch mi c th. So snh in di san phm PCR ca nhiu ging c c im khc nhau trong cng loi, vi nhiu primer khc nhau c th gip xc nh ban gen ca sinh vt. Hin nay, ngoi RAPD cn c nhiu loi chi th phn t (molecular marker) khc a c pht trin nh a hnh chiu di cc on ct hn ch (restriction fragment

length polymorphism, RFLP), vi v tinh (microsatellite) hay cn gi l s lp li cc on nucleotide n gian (simple sequence repeats, SSR), v a hnh chiu di on DNA c khuch ai chn lc (amplified fragment length polymorphism, AFLP). Cc k thut ny c xy dng trn c s PCR (ngoi tr RFLP) c trin vng rt ln trong nghin cu da dng di truyn (genetic diversity), nh v gen (gene location) v lp ban gen do chng n gian v mt k thut, tit kim thi gian v c hiu qua cao. PCR hin nay con c dng thay th cho in di isozyme phn loi thc vt, xc nh mi quan h ca chng mt cch chnh xc.

4. ng dng trong chn on lm sng Trc y, k thut phn tch RFLP thng c s dng xc nh cc t bin dn ti cc bnh di truyn ngi. Tuy nhin, k thut ny ch p dng c trong trng hp t bin dn n s thay i trnh t c th pht hin c trn c s di ca on DNA. Ngc li, trng hp mt s t bin gy ra bnh di truyn nhng khng to ra cc on DNA ct hn ch khc nhau khi phn tch RFLP th ch c th pht hin nu xc nh c trnh t on DNA cha im t bin. Nh vy, chng ta buc phi xy dng th vin h gen (xem chng 5) cho mi ngi, sau to dng v xc nh trnh t nucleotide ca dng gen t bin, v th phng php ny i hi tn nhiu thi gian. K thut PCR cho php a ra mt chn la khc n gin hn cho php thu nhn thng tin v trnh t gen rt nhanh bng cch khuch i on gen cha im t bin v sau phn tch trc tip cc sn phm PCR thu c. Kh nng nhn dng nhanh cc t bin khng ch quan trng trong chn on lm sng, m cn y nhanh vic nghin cu cc bnh di truyn. nhy cao ca k thut PCR cho php ng dng d dng trong cc chn on bnh nhim trng. V d: vic khuch i mt s lng ln DNA virus trong cc mu bnh cho kh nng chn on bnh sm hn trc khi triu chng bnh bt u xut hin. iu ny gip cho thy thuc c phc iu tr bnh thch hp, c bit cc bnh ung th do virus gy ra (v d: ung th vm hng do papillomavirus ngi gy ra) v thng thng c kt qu hn nu nh bt u iu tr giai on sm.

Tai liu tham kho/c thm

1. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA and Struhl K. 2002. Short Protocol in Molecular Biology. Vol 1 and 2. 5th ed. John Wiley & Sons, Inc. USA. 2. Chen BY and Janes HW. 2002. PCR Cloning Protocols. 2nd ed. Humana Press Inc. New Jersey, USA. 3. Dieffenbach CW and Dveksler GS. 2003. PCR Primer: A Laboratory Manual. 2nd Edition. Cold Spring Habor Laboratory Press, NewYork, USA.

4. Innis MA, Gelfand DH, Sninsky JJ and White TJ. 1990. PCR Protocols: A Guide to Methods and Applications. Academic Press, New York, USA. 5. Maniatis T, Fritsch EF and Sambrook J. 1989. Molecular Cloning-A Laboratory Manual. Cold Spring Habor Laboratory Press, USA. 6. Rapley R and Walker JM. 1998. Molecular Biomethods Handbook. Humana Press Inc. New Jersey, USA. 7. Surzycki S. 2000. Basic Techniques in Molecular Biology. Springer-Verlag, Berlin, Heidelberg, Germany. RAPD (random amplified polymorphic DNA): DNA a hnh c khuch i ngu nhin . STS (sequence-tagged site): STS primer c thit k trn c s cc trnh t ca RAPD markers.
1 2

Chng 4

Cac h thng vector

Co ba nhm vector chnh c s dung trong tao dong DNA va nhn ban chung trong t bo vt ch (E. coli hoc nm men), bao gm: 1) Nhm plasmid, 2) Nhm phage/phagemid, v 3) Nhm nhim sc th nhn to (artificial chromosome: BAC v YAC). Y tng v vector chuyn gen bt ngun t cac plasmid cua vi khun. Vector chuyn gen la phn t DNA co kha nng t tai sinh, tn tai c lp trong t bao va mang c cac gen cn chuyn. Cac vector chuyn gen phai thoa man cac yu cu ti thiu sau: - Co trinh t khi u sao chep (ori) co th t sao chep ma tn tai c lp. - Co cc v tr nhn bit n (single recognition sites-SRS), ni ma cac enzyme han ch nhn bit ct ri lam ch lp rap oan gen ngoai lai vao. Cac trinh t nay thng nm xa im khi u sao chep tranh bi ct nhm. - Co trinh t khi ng (promoter) tin hnh phin ma gen ngoi lai. - am bao s di truyn bn vng cua DNA tai t hp dang c lp hay gn vao nhim sc th cua t bao vt chu. - Co cac gen ch th (marker genes) d dang phat hin ra th ti t hp. Ngoai ra, chung con phai co nhng c tinh b sung khac cho vic tao dong d thc hin nh l: - Cha cac gen lam v hiu hoa oan DNA khng mong mun c gn vao. - Co nhiu ban sao c tch ra khoi t bao vi s lng ln va am bao s khuch ai cua gen ngoi lai.

- Ngoai promoter cn co thm cac trinh t nucleotide khac cn thit cho s biu hin cua gen, chng han nh: RBS (ribosome binding sites-vng lin kt vi ribosome dich ma). Gia tri cua vector ch no c thit k sao cho tht thun tin vi cc muc ich s dung khc nhau. Khng co vector toan nng cho chuyn gen, ma cn co s chon la tuy mc ch v kich thc ca oan gen c tao dong. Chung c thit k vi nhiu chc nng chuyn bit mang c cac trinh t nucleotide nh mong mun. Cac vector chuyn gen co nm ng dung quan trong chu yu: - Tao dong (cloning) va khuch ai (amplification) mt on trinh t cua DNA (nhiu ban sao ging nhau). - Nghin cu s biu hin cua mt oan trinh t DNA. - a gen vao cac t bao vi sinh vt hay cac ng-thc vt . - San xut RNA. - San xut protein t gen c tao dong. Chng ny gii thiu su loi vector ph bin (thuc ba nhm vector: plasmid, phage/phagemid v nhim sc th nhn to) dng trong to dng gen (gene cloning): plasmid, bacteriophage , cosmid, bacteriophage M13, BAC v YAC. I. 1. c im cua plasmid Plasmid vector

Plasmid la cac thng tin di truyn ngoai nhn, c tim thy trong nhiu loai vi khun khac nhau. Chung la nhng phn t DNA mach vong, si i, kich thc t 1- 200 kb, co kha nng t sao chep tn tai c lp trong t bao. Cac plasmid co th mang mt s gen cua vi khun va cac gen nay co th biu hin ra protein. Mt s kiu hnh khc nhau ca plasmid nh: - Khang cac cht khang sinh - Khng kim loi nng - San xut cac cht khang sinh - Thuy phn cac hp cht hu c phc tap - San xut c t ng rut enterotoxin - Sn xut cht dit khun bacteriocin - San xut cac colicin1 - Sn xut haemolysin2 - San xut cac enzyme sa i3 va enzyme han ch 4. Mt loi vi khun vt ch thng cha hai hoc nhiu loi plasmid cng tn ti. Chng c th l cc plasmid tng hp (compatible plasmid) hoc khng tng

hp (incompatible plasmid). Ngi ta c th chia plasmid lm hai loi da trn c im sinh sn v phng thc sng ca chng, l: plasmid tip hp (conjugative plasmid) v plasmid khng tip hp (non-conjugative plasmid). Trong t nhin, plasmid xm nhp vao t bao vt chu nh s tip hp (conjugation) cua vi khun, nhng trong nghin cu thc nghim cac plasmid c chuyn nap vao vi khun bng mt quy trinh nhn tao goi la bin nap gen (gene transformation). Kiu hinh mi cua th nhn (recipient) do plasmid em n (Vi du: plasmid khang khang sinh) cho phep chon loc xac inh cac nhn t bin nap va duy tri plasmid trong qun th vi khun. cac vi tri ct han ch ca plasmid, DNA ngoai lai (foreign DNA) co th chn vao va c xac inh vi gen ma hoa cho cac marker chon loc. Mt trong cc v ector hay c s dung trc y la pBR 322 mang hai gen khang r r ampicillin (Amp ) va tetracycline (Tet ) cung mt s vi tri ct han ch thun li. Co hai dang phn chia t pBR 322 thich hp cho s tai ban la cac vector pAT 153 va pXf 3. Cac plasmid kich thc nho co s lng ban sao ln hn va mn cam hn vi vi khun. Tuy nhin, vic giam kich thc cua plasmid co th lm mt cac vi tri tao dong (multiple cloning sites, MCS) hu ich. Vi du : pXf 3 thiu cac vi tri BalI va AvaI (cac vi tri nay co trong pBR 322 va pAT 153). Mt s plasmid khng c s dung rng rai nh pBR 322, ma chi c s dung trong cac muc ich c bit, vi du: - pMK 16: cha cac vi tri nhn bit n SmaI va XhoI trong gen ma hoa tinh khang kanamycin. - pKC 7 va pACYC 184: mang cac vi tri tao dong hu ich va gen ma hoa tinh khang chloramphenicol. - Cac plasmid c kch thc ln pCR1 (11,4 kb) va pSC 101 ( 9,9 kb): khng c dung thng xuyn trong cac thi nghim tao dong. Thng thng, s sao chep DNA plasmid c tin hanh nh cac enzyme tai ban 5 nhim sc th vi khun. mt s plasmid dng stringent control s sao chep cua chung kh t hoc b gii hn, ch gp i s sao chep cua t bo vt chu va chi mt hoc mt s ban sao cua chung co mt trong mi t bao vi khun. Cac plasmid dng 6 relaxed control co s lng ban sao trong mi t bao nhiu hn stringent plasmid, khoang 10-20 va co khi ln n hang ngan bn sao nu nh s tng hp protein bi dng lai (Vi du: bng cach x ly chloramphenicol). Nu thiu s tng hp protein thi s sao chep cua cac relaxed plasmid c tip tuc, trong khi s sao chep cua DNA nhim sc th va cua cac stringent plasmid bi dng lai. Cac plasmid dung lam vector tao dong phai co mt s tinh cht sau: - Kich thc tng i nho va phai tai ban trong dng relaxed plasmid. - Mang mt hoc nhiu marker chon loc (selectable marker) cho phep xac inh cac nhn t bin nap va duy tri plasmid trong qun th vi khun.

- Cha cac vi tri nhn bit n cho mt hoc nhiu enzyme han ch. n nay, cac plasmid a c cai tin ngay cang thun tin hn cho ky thut tai t hp DNA, tri qua ba th h: - Th h u tin. La cac plasmid t nhin n nay hu nh khng con s dung na. - Th h th hai. La cac plasmid c cu tao phc tap hn. Mt trong nhng plasmid thng c s dung la pBR 322 (Hinh 4.1) co ngun gc t mt plasmid nho ColE1. No c thit k t nhiu oan cua cac plasmid khac nhau va co cac gen r r khang thuc (Amp va Tet ), trinh t khi u sao chep (ori), va co cac v tr nhn bit r cho cac enzyme han ch nh Eco RI , Hin dIII , Bam HI , Sal I ,... oan gen Amp co r bn im nhn bit, gen Tet co tm im nhn bit, nhng im nay giup d phat hin cac plasmid co gen ngoai lai gn vao. Vi du : nu ct plasmid bng enzyme BamHI (375) r ri gn DNA ngoai lai vao ch ct thi gen Tet bi phn i do chn oan DNA la, v th t bao mt kha nng khang tetracycline nhng vn khang ampicillin. Plasmid nay co kha nng sao chep c lp vi t bao E. coli va tn tai vi s lng trung binh 20-30 ban sao cho mi t bao. Trong nhng iu kin nui cy nht inh co th khuch ai co chon loc lam tng s plasmid n hn 1.000 ban sao cho mt t bao.

Hinh 4.1. Plasmid vector pBR 322. Apr (hay Ampr) v Tetr: gen khng ampicillin v tetracycline, ori: trnh t khi u sao chp, v mt s v tr nhn bit cho cc RE. - Th h th ba . L a cac plasmid a nng (polycloning plasmid) va chuyn dung. tin cho vic s dung nhiu lo ai RE khac nhau, nhiu trinh t nhn bit cua chung c xp ni tip nhau thanh mt oan dai goi la polylinkers (vng a ni) hay multiple cloning sites (cac vi tri tao dong). Cac plasmid vi khun co th cha oan DNA ngoai lai

khoang 3-10 kb. Co 3 nhom plasmid thng dung hin ang c ban rng rai trn thi trng nh: + Nhom cac plasmid pUC. Kich thc khoang 2.600 bp, mang gen Apr va mt phn gen lacZ, xen vao gia gen lacZ la polylinker (Hinh 4.2). Cac thanh vin cua nhom nay chi khac nhau do dai va chiu cua polylinker. Cac u im cua nhom nay: * Kich thc nho. * S co mt cua gen lacZ 7 rt thun tin cho vic phat hin vector tai t hp. * Vung polylinker cho phep chn vo gn nh bt ky mt trinh t DNA la nao.

Hinh 4.2. Plasmid vector pUC19 . V tr to dng (t 396-447) c gn vo gen lacZ, nhng khng can thip vo chc nng ca gen. + Nhm cac plasmid pGEM : Kich thc khoang 3.000 bp, mang gen Ampr, gen lacZ, polylinker v promoter c trng cho cac RNA polymerase (SP6, T7) hai bn vung polylinker cho phep phin ma oan DNA gn trong vector thanh nhiu RNA (Hinh 4.3). Cac RNA nay thng dung lam probe (mu do) hay dung trong nghin cu cu truc va chc nng cua RNA.

Hinh 4.3. Plasmid vector pGEM. Nhm vector ny c m vng sn mang 2 u T vng MCS, c trng cho vic gn cc sn phm PCR mang 2 u A. + Nhm cac plasmid pBluescript : Kich thc khoang 3 . 000 bp, cac plasmid nay kt hp c tt ca nhng u im cua my nhom va k va c xem la nhom co tim nng ng dng ln nht hin nay (Hinh 4.4). 2. Tao dong trong plasmid V nguyn tc, DNA plasmid bi ct bi enzyme hn ch va gn in vitro vi oan DNA ngoai lai tao ra cac plasmid tai t hp, sau o chung c dung bin nap vao vi khun. Kho khn ln nht la phn bit gia cac plasmid cha cac oan DNA ngoai lai th tai t hp (recombinant) va cac phn t DNA vector a tai tao lai vong (recircularization). S tai tao lai vong cua plasmid co th c han ch bng cach iu chinh nng DNA ngoai lai va DNA vector trong phan ng gn (ligation). Mt s phng thc c dung phn bit gia cac th tai t hp va tai tao lai vong nh sau:

Hinh 4.4. Plasmid vector pBluescript II SK (+ /- ). Vector ny mang vng to dng v tr t 598-826 trn gen lacZ, gen khng ampicillin, cc promoter T3 v T7, v cc v tr gn cho cc cp primer khc nhau dng trong phn tch trnh t on DNA ngoi lai. 2.1. Kh hoat tinh bng chn oan (insertional inactivation) Phng phap nay dung cho cac plasmid mang hai hoc nhiu marker (v d: Ampr, lacZ). DNA ngoai lai va DNA plasmid c thuy phn cng mt loi enzyme hn ch va tinh sach, sau o gn hai DNA vi nhau, hn hp gn c dung bin nap vao E. coli mn cam Amp chon loc th bin nap nh tinh khang Amp cua plasmid v hot tnh b -galactosidase ca gen lacZ. Cc khun lac cha cac plasmid tai t hp sinh trng trn mi trng co mt Amp v isopropyl-thiogalactoside (IPTG) cng vi c cht nhim sc th X-gal s c mu trng do on DNA ngoi lai xen vo gia gen lacZ lm gen ny mt hot tnh. Trong khi cc khun lac cha DNA plasmid tai tao lai vong s c mu xanh do gen lacZ khng b mt hot tnh (Hnh 4.5 v 4.6).

Hnh 4.5. C ch tc dng ca -galactosidase 2.2. Tao dong inh hng (directional cloning) Hu ht cac plasmid vector mang hai hoc nhiu vi tri nhn bit enzyme hn ch, vi d: vector pBR 322 cha cac vi tri nhn bit n HindIII va BamHI sau khi ct bng hai enzyme hn ch tng ng, oan DNA plasmid ln hn co th c tinh sach bng in di agarose gel va gn vi mt oan DNA ngoai lai mang cac u kt dnh tng ng vi no cung c ct bi BamHI va HindIII. Kt qua, th tai t hp dang vong mang tinh khang Amp sau c dung bin nap vao E. coli. Do thiu s b tr gia cac u li HindIII va BamHI nn oan vector ln hn khng th ti tao li vong mt cach hiu qua c vi th no bin nap vao E. coli rt km. Di nhin, cac t hp khac cua enzyme cung co th c s dung tuy thuc vao cac v tr nhn bit (RS-recognition sites) trong vector va ca oan DNA ngoai lai (Hnh 4.7).

Hnh 4.6. To dng theo phng thc kh hot tnh bng chn on. Amp r : gen khng ampicillin, lacZ: gen m ha enzyme -galactosidase.

Hnh 4.7. To dng nh hng. Tet r : gen khng tetracycline, Tets : gen khng tetracycline b khuyt on v mt hot tnh. 2.3. Bin np vector ti t hp vo vi khun/t bo vt ch Sau khi to c vector ti t hp mang gen ngoi lai, vic tip theo l bin np n vo t bo vt ch. Trong trng hp ny t bo vt ch thng c s dng l vi khun E. coli khuch i mt lng ln DNA plasmid ti t hp dng cho cc phn tch v sau.

Hai phng php c dng bin np vector ti t hp vo E. coli l in bin np (electroporation transformantion) v ha bin np (chemical transformation). 2.3.1. in bin np y l k thut hiu qu nht bin np vi khun. Hai thng s quan trng ca phng thc ny l loi t bo vi khun v tn s xung in cn thit. Th tch ca dung dch t bo thng c dng l 30 L (tng ng vi nng 1010 t bo E. coli/mL) c b sung 5 ng plasmid trong mt cuvette c khong trng in cc (electrode gap) 0,1 cm. Hiu sut bin np ca phng thc ny ln hn 110 9 th bin np/g plasmid siu xon v khong 1108 th bin np/g plasmid c dng trong phn ng gn. Tn s bin np khong 0,02 cho c hai loi plasmid. Tn s bin np thp ngn cn c s ng bin np (co-transformation) vo vi khun ca hai hoc nhiu phn t plasmid. Phng php in bin np c mt s u im sau: - Hiu sut bin np cao. - C th dng mt th tch dch t bo nh. Th tch dch t bo khong 20 L c th cho hiu sut khong 109 th bin np. - Phng php chun b t bo bin np rt n gin, khng s dng cc k thut phc tp v tn thi gian. Hn na, cc t bo dng bin np c th c chun b trc v bo qun v hn nh m khng mt tnh kh bin. - Tn s in bin np vi DNA siu xon v DNA mch vng l ging nhau. Do , khng cn thit phi dng vector c tinh sch cao trong cc phn ng gn. - Hiu sut bin np phn t cho DNA mch vng rt cao i vi cc plasmid c kch thc ln n 50 kb. Tuy nhin, nhc im ca phng php ny l i hi thit b bin np t tin. 2.3.2. Ha bin np y l phng thc kinh in bin np DNA plasmid vo t bo E. coli. Cc t bo c trong dung dch CaCl2 tr thnh t bo kh bin gip cho chng d tip nhn DNA plasmid. DNA plasmid a vo bng cch shock nhit nhanh (40-50 giy), cc t bo bin np sau c chn lc bng phng php chn lc dng tnh trn a agar cha mi trng LB vi khng sinh thch hp. Mi khun lc trn a khng sinh i din cho mt th bin np n. Cc t bo cha plasmid mang DNA ngoi lai c th xc nh bng mt trn a mi trng c b sung thm c cht nhim sc th cho -galactosidase (X-gal) v chng l cc khun lc khng mu do s kh hot tnh ca enzyme bng cch chn on DNA ngoi lai.

Phng php chun b v bo qun t bo kh bin trong ha bin np cng rt n gin. Hiu sut bin np ca phng php ny trong khong 104-106 th bin np/g plasmid, ty thuc vo kch thc ca on DNA chn (insert DNA) v chng vi khun c s dng. Hiu sut ny thch hp cho cc phng thc to dng truyn thng. i vi cc phng thc cn hiu sut bin np cao hn (v d: xy dng th vin cDNA, xy dng th vin phn tch trnh t DNA...) th tt hn ht l dng phng php in bin np. Tuy nhin, nu khng c sn thit b bin np bng in th vn c th thu c hiu sut bin np cao bng cch dng cc chng vi khun thch hp hn cho mc ch ny v c th thu c hiu sut 10 9 th bin np/g plasmid. II. Bacteriophage vector Phage l la mt virus mang DNA si i co kich thc khoang 50 kb, DNA cua no dang phn t mach thng co hai u b tr si n dai 12 nucleotide (c dung lam u cos). Sau khi xm nhim vao vi khun vt chu, DNA tao vong ngay lp tc bng cach ghep hai u kt dinh cos nh enzyme DNA ligase cua vt chu (Hinh 4.8). Cac giai oan tip o co th dn n vic hoc lam tan (lysis) t bao vt chu (bao gm vic san xut va giai phong cac phage th h con) hoc la gy ra hin tng tim tan (lysogeny) (trong giai oan nay DNA hp nht vao trong chromosome cua vt chu). Cac phage co kha nng thc hin tai nap mang gen t t bao vi khun cho (donor) sang t bao nhn (recipient). Vector phage c s dung rng rai xy dng th vin gen (gene library), vi no mang c oan DNA ngoi lai ln hn (15-23 kb) so vi plasmid, d bao quan, d tach ra phn tich. u im ni bt cua cac phage la chung co h thng t ng xm nhp va sinh san trong t bao vi khun vi mt hiu qua cao hn nhiu so vi vic a plasmid vao t bao vi khun bng bin nap. Tuy nhin cac thao tac ban u co phc tap hn.

Hinh 4.8. Ban n gian cua bacteriophage genome. Cc gen lin quan n cc chc nng khc nhau c trnh by trn s . cIII, N, cI, cro v cII: cc gen lin quan n hot ng iu ha, min dch tin phage, siu nhim. O v P: cc gen tng hp DNA. Q: gen iu ha chc nng mun. S v R: cc gen phn gii t bo vt ch. PL: promoter bn tri, PR: promoter bn phi, L-cos: u kt dnh bn tri, R-cos: u kt dnh bn phi. 1. c im cua bacteriophage Trc y, ngi ta nghi rng vic s dung phage lam vector la rt bt tin do DNA cua no qua dai (50 kb), mun ct ra nhiu oan thi phai co nhiu trinh t ct. Tuy nhin, v sau ngi ta a phat hin gia DNA cua phage co vung m (stuffer) hay con goi la vung trung tm c chiu di bng khong 1/3 chiu di ca phage , vung nay khng quan trong va nu ct no i thi vn khng anh hng n kha nng sinh san cua phage l trong t bao vi khun chu va kh nng lam tan t bao chu. V th, ngi ta a ct bo on stuffer lp cac oan gen ngoai lai vao. Do DNA phage co dang mach thng nn khi bi ct gia se tach ri lam thanh hai nhanh (arms): phai va trai. oan gen ngoai lai c gn vao gia sao cho DNA tai t hp khng ln hn 105% hay nho hn 78% cua b gen binh thng cua phage . Kha nng co th mang mt DNA ngoai lai dai hn cua plasmid la mt u th cua phage . iu o co nghia rng khi ly oan stuffer ra (chi con 60% chiu dai binh thng cua phage ) thi no qua ngn lp vao u, no chi lp c khi DNA ngoai lai gn thm vao ch trng. Nh vy, d xac inh DNA ngoai lai a gn vao phage hay cha va ng thi tinh cht nay cung giup loai bo cac phage khi oan DNA ngoai lai bi ct ri ra. Trn vector phage ngi ta cng a chon c cac v tr nhn bit cho cac enzyme ct han ch, va xy dng phng phap ong goi in vitro (in vitro packing) a oan DNA a c gn vao u cua phage. 2. Tao dong trong bacteriophage Bacteriophage vi genome phc tap va ln c dung nh mt vector, DNA cua no cha mt s vi tri thich hp cho cac RE cn thit tao dong. Cac bc tao dong trong phage c tm tt nh sau (Hnh 4.9 v 4.10): - Tinh sach DNA vector bng ly tm theo gradient tc hoc in di gel sau khi a c ct bng RE. - Cac nhanh phai va trai sau o c lin kt vi DNA ngoai lai kch thc khong 20 kb co u kt thuc tng ng vi u cua cac nhanh. - Kt qua cac DNA tai t hp c ong goi in vitro trong cac phage . - Cac phage tai t hp mang cac on DNA ngoai lai mong mun c xac inh bng phng php lai nucleic acid.

- Tinh sach va chon t bao vt chu ( E. coli) cho no sinh san duy tri va phn tich cc oan DNA ngoi lai. 2.1. Chon vector thich hp Co hai yu t anh hng n s chon loc, l enzyme ct hn ch c s dng v kich thc DNA ngoai lai. Chi khoang 60% genome (nhanh trai v nhanh phai) la cn thit cho s sinh tan cua phage. Kha nng sng sot cua phage giam khi DNA dai hn 105% hoc ngn hn 78% so vi genome t nhin a c ong goi. iu quan trong la chon vector va DNA ngoai lai nh th nao kich thc cua phage tai t hp nm trong gii han cho phep.

Hnh 4.9. Cc bc to dng trong bacteriophage . DNA ngun c ct bng BamHI v c phn on theo kch thc (khong 20 kb). Bacteriophage l cng

c phn ct bng BamHI. Hai mu ny c trn vo nhau v x l bng T4 DNA ligase. Phn ng gn xy ra to mt bacteriophage mi (ti t hp): on stuffer c thay bng on DNA ngoi lai. Nhng phn t ny ng gi in vitro trong u ca phage , v chng c th gy nhim sau khi c thm phn ui

Hnh 4. 10 . Phn t DNA ca phage . A: T ti bn DNA t dng vng ca lm cho n tr thnh dng dy thng, hnh thnh cc on chng lp ln nhau, vi di khong 50 kb. B: Mi u u cha y 50 kb n v ca l DNA , trc khi ui c tng hp gn vo sau. Mt s vector a c thit k thun li cho sinh trng cua chng trong E. coli + mang prophage P2, phenotype nay c goi la Spi . Cac chung thiu hai gen cn thit trong tai t hp (red va gam) biu hin phenotype Spi se sinh trng tt trong cac th tim tan P2 chng nao ma chung con mang chui chi (chi la c cht cho s tai t hp c xuc tac bi h thng recA). Tao dong cc vector L47.1, 1059 hoc BF101 a c tin hanh bng cch loi b oan stuffer cha red va gam. Kt qua thu c phage tai t hp la Spi - va chung co th c nhn bit hoc chon loc bng kha nng sinh trng cua chung trn + cac th tim tan P2 recA cua E. coli . Hu ht cac vector thay th mt gen gam khi oan stuffer bi loi b. Cac th tai t hp c tao thanh vi cac vector nh th phai c sinh san trong vi khun recA+. Phage l sep6-lac5 mang cac gen red va gam trong nhanh phai cua no v cac th tai t hp + c tao thanh vi vector nay c phenotype Spi co th c nhn ln trong vi khun mang t bin recA .

2.2. Nhng vector c ci tin Nhng vector thng c ci tin nhm mc ch: - Tng kh nng thch ng ca cc on DNA ngoi lai khc nhau vi cc RE. - Cho php chn la phng thc ti t hp mong mun. - Cho php c c cc RNA probe sn sng cho vic chuyn m ca DNA ngoi lai gn vo vector. iu ny gip chng ta d dng sng lc cc th vin trong qu trnh chromosome walking, v d nh ZAP. - Pht trin cc vector trong qu trnh gn vo cDNA ca sinh vt bc cao, di hnh thc mt polypeptide dung hp vi -galactosidase. Hnh thc ny rt hu ch trong vic chn lc khng th, v d nh vector gt11.

Hnh 4.11. Vector EMBL3 v EMBL4 c thit k t phage l mang cc v tr nhn bit cho cc enzyme SalI, BamHI v EcoRI Th h gn y nht ca vector l l EMBL3 v EMBL4 (Hnh 4.11), chng mang cc trnh t c tnh cht polylinker nm gn on c th thay th c. Cc phage vi nhng th insert nh bn trong n c th c chn lc bng kiu hnh Spi -, chnh l chi+. Th ci tin t EMBL3 c nhng t bin EMBL3 Sam, EMBL3 Aam Sam. Nhng t bin trong vector khng ch lm gia tng kh nng cha ca n, m cn c s dng trong h thng chn lc nhng trnh t DNA c phn lp, lin kt vi cc gen c ch (suppressor).

III . 1. c im ca cosmid

Cosmid

vector

Cosmid la cac vector c bit c xy dng bi plasmid + u cos dng tao dong cac oan DNA kch thc ln cua eukaryote (Hnh 4.12). Cac thanh phn khng th thay th cua cac cosmid vector bao gm: - Cc marker khang khng sinh va mt trnh t ori cua plasmid. - oan DNA mang u dinh cos cua phage l . - Kich thc nho sao cho cac oan DNA eukaryote dai khong 45 kb co th thich ng.

Hnh 4.12. Bn v vng to dng ca vector cosmid pWEB-TNC. V tr to dng SmaI nm bn cnh cc cp BamHI, NotI v EcoRI. Chlr: gen khng chloramphenicol. 2. To dng trong cosmid Cac nguyn tc c ban cua phng phap tao dong trong cosmid c trinh bay hinh 4.13. Trc ht cosmid c ct vi RE th nht ( ScaI) to thnh mch thng, sau phn t mch thng c ct tip vi RE th hai (BamHI) lm thnh hai nhnh (nhnh ln v nhnh nh). Mt nhnh cha u cos, gen khng khng sinh v vng ori; nhnh cn li ch cha u cos th hai. Hai nhnh ny kt hp vi genomic DNA c ct vi RE (cng loi vi RE th hai ct cosmid thnh hai nhnh) nh

DNA ligase, cui cng tt c chng c ng gi in vitro trong u ca cc tiu th phage l trng thnh. S ong goi cac phn t tai t hp trong cac tiu th phage ch thc hin cho cac th tai t hp co kich thc khoang 40-50 kb. Cac cosmid vector c thit k ti u nht co chiu dai t 4-6 kb va vi th chung co th thich hp vi DNA ngoai lai c kch thc khong 45 kb. Mc du co nhng thun li v kich thc, cac cosmid dung lam vector cung co mt s kho khn sau: - Gn vector vi vector: s tai t hp trong phn t (intramolecular) gia hai hoc nhiu vector trong mt cosmid n co th dn n ct bo DNA cua cosmid vector va cui cung mt cosmid do phn ly. - S cht ng ln xn (scrambling): xut hin do s xm nhim vao trong cung mt vector cua hai hoc nhiu oan khng canh vi oan DNA mong mun trong b gen gc (original genome). - Kho khn trong vic sang loc mt s lng ln khun lac vi khun. S tin li cua phng phap tao dong cac oan DNA ln trong cosmid a c Royal va cs. (1979) cng b u tin. ng co th phn lp t th vin cosmid cua DNA ga hai oan chng ln nhau (overlapping) mang gen ovalbumin va hai gen ging ovalbumin. Gn y hn, cac th vin gen dng cosmid vector mang DNA rui gim, DNA chut, DNA ngi cung a c xy dng. Hnh 4.13 cho thy cosmid c cha v tr ori ca E. coli (cho php cosmid c duy tr nh mt plasmid trong E. coli). Hai u cos gn v tr ct hn ch ScaI v BamHI. DNA ngun c ct bi BamHI phn on c kch thc khong 40 kb. Gen khng Tet (Tet r ) nh v gn cos. Phn t dng plasmid c ct bi BamHI v ScaI. Ba mu DNA ny c trn vo nhau v c gn bng T4 DNA ligase. Sau khi gn xong, nhng phn t ny c ng gi trong phn u ca phage l , v nhng phn t c th ly nhim s c hnh thnh sau khi to ui.

Hnh 4.13. To dng trong cosmid IV. Bacteriophage 1. c im ca bacteriophage M13 M13 vector

M13 la phage dang si (filamentous bacteriophage) vi DNA vong ong, dai khoang 6.500 nucleotide (Hnh 4.14). M13 c xem nh l mt vt truyn tao dong (cloning vehicle), cac vector M13 va cac DNA primer c bit a c xy dng cho

phep tao dng on DNA ln hn 350 bp t mt dong n. Tao dng cac oan DNA dai c tin hanh bng cach tao dng tng on ngn chng ln nhau. 2. To dng trong bacteriophage M13 Vn chinh trong tao dong M13 la s khng n inh cua cac oan DNA ngoai lai c kch thc ln, cac trnh t ln hn 1.000 nucleotide thng khng n inh va lam tng cac oan khuyt trong qu trnh sinh san cua bacteriophage. Tuy nhin, cac oan DNA ngoai lai gm 300-400 nucleotide thng la n inh tin hanh cac ng dung nh a noi trn. Ngoai tr vung 507 nucleotide c bit nh la chui lin gen (intergenic sequence-IS) con tt c h gen cua M13 u cha thng tin di truyn khng thay th cho s sao chep cua virus. Vung IS nhn cac oan DNA ngoai lai nhng khng anh hng n kha nng sng sot cua t bao. Trong cac dang phn chia cua M13 co hai chui (trnh t) xm nhim: - u tin la chui operon lac cua E. coli cha gen iu hoa (regulator) va ma hoa thng tin cho 146 amino acid u tin cua gen -galactosidase ( Z). Phn amino u tn cung cua protein -galactosidase co th b tr (b tr ) gen b -galactosidase sai hong (defective) co mt trong episome F trong t bao vt chu. S b tr nay san xut -galactosidase hoat ng lam tng mau xanh khi phage va cac t bao sinh trng trong mi trng co nhn t cam ng isopropyl-thiogalactoside (IPTG) va c cht nhim sc th X-gal. - Loai chui th hai la oan DNA nho, polylinker c cha mt s vi tri ct han ch a c gn trong u tn cung amino cua gen -galactosidase. S xm nhim nay khng anh hng n kha nng b tr cua chui peptide -galactosidase cho t bin galactosidase. Cac phage mang cac oan chn lam tng cac vt khng mau khi sinh trng trn mi trng co mt IPTG va X-gal.

Hnh 4.14. Dng t nhin ca bacteriophage M13. Cc gen t 1-10 c cc chc nng khc nhau lin quan n cu trc v protein v pht sinh hnh thi ca phage. Vector tao dong M13 ung tiu chun (phagemid) c trinh bay hinh 4.15, cac vector tao dong M13 khac loai vector k trn l cac vi tri ct han ch trong polylinker.

c bit, cac oan DNA nho co vai tro nh mt primer cung c tao dong trn plasmid hoc c tng hp hoa hoc. Cac primer nh vy tng ng vi vector M13 trong oan gen b -galactosidase canh oan polylinker.

Hnh 4.15. Vector M13mp18 (phagemid). y l mt trong cc vector c s dng ph bin ca nhm vector M13mp. V. 1. c im ca BAC BAC vector

H thng nhim sc th nhn to ca vi khun (bacterial artificial chromosomeBAC) da trn c s plasmid F-factor, n c th ti bn trong E. coli vi cc on chn c kch thc ln n 300 kb. Th ti t hp BAC c bin np vo trong t bo vi khun bng xung in (electroporation)8. Cc BAC thun li cho vic xy dng th vin, lp bn v phn tch genome. Chng c hiu sut to dng cao, cc on chn DNA c thao tc d dng v duy tr n nh. Mt plasmid F c bn bao gm bn vng cn thit c chc nng n nh plasmid v quyt nh s lng bn sao. l parA, parB, oriS v repF. C hai parA v parB u cn cho vic phn on v n nh plasmid. Ngoi ra, parB cn cn cho vic kt hp vi cc F-factor khc, oriS l gc ti bn DNA, repF mang tn hiu ca protein E cn cho qu trnh t ti bn t oriS v qu trnh kim sot s lng bn sao. Trn plasmid F ngi ta cn b sung thm gen khng chloramphenicol (CMr hoc Chlr) v

gen lacZ lm marker chn lc cc th bin np. Chng E. coli c s dng ph bin cho k thut to dng BAC l DH10B, c im chnh ca n l mang nhng t bin ngn cn: - S phn ct DNA l bi h enzyme ni sinh (hsd/RMS). - S phn ct DNA c gc methyl (5-methyl cytosine, 5-hydromethylcytosine, gc methyladenine) (mcrA, mcrB, mcrC v mrr). - S ti t hp (recA1). Nhn chung, cc BAC vector c cc c im tng t vi cc plasmid vector tiu chun ca E. coli, nh : - Cha vng ori cn thit cho ti bn ca plasmid. - C ngun gc t mt plasmid ln trong t nhin: F-factor. - C s lng bn sao thp (1-2 bn sao/t bo). - Hiu sut bin np thp c khc phc bng phng php xung in a DNA ti t hp vo t bo E. coli. Mt s u im ca h thng BAC so vi YAC: - n nh. - D bin np. - Tc sinh trng ca vt ch E. coli nhanh. - D tinh sch. V th, hu ht genome ngi u c phn tch trnh t bng cch dng cc dng BAC hn l cc dng YAC. 2. To dng trong BAC Qu trnh to dng trong BAC vector c trnh by trong hnh 4.16, bao gm cc bc sau:

Hnh 4.16. To dng trong BAC vector - BAC vector c ct bng enzyme hn ch HindIII (c v tr nhn bit nm trn gen lacZ), sau hai u HindIII ca vector c dephosphoryl ha bng phosphatase ngn cn s ti to li vng.

- DNA c khi lng phn t cao c bc trong agarose v sau phn on cng bng HindIII nh trng hp vector. - Gn vector v cc on DNA c kch thc >150 kb bng enzyme T4 DNA ligase, sau bin np vo E. coli bng xung in. - Chn lc th ti t hp trn mi trng nui cy c b sung CM v IPTG. Cc khun lc c mu trng l khun lc mang th ti t hp, cn cc khun lc c mu xanh l khun lc khng mang th ti t hp. VI. 1. c im ca YAC YAC vector

Nhim sc th nhn to ca nm men (yeast artificial chromosome-YAC) l cc vector to dng mch thng da trn cu trc nhim sc th t nhin ca nm men. Chng c th c ct thnh hai on hoc hai nhnh nhim sc th nhn cc on rt ln c kch thc ln n 2.000 kb. Cu trc ca YAC sau c th a vo cc t bo nm men c thnh t bo b loi b (spheroplast), chng c duy tr n nh nh cc nhim sc th t tr (autonomous). Th vin YAC c lp rp v s dng cho vic lp bn cc vng c kch thc ln ca DNA ngi. Do kch thc ln ca chng cho nn k thut phn tch thch hp hn c l in di gel trng gin on (pulsed field gel electrophoresis-PFGE). Nhc im ca YAC l hiu sut to dng ca mt vi h thng tng i thp, s xut hin cc dng khm (cc t bo bin np cha hai mu DNA khng lin k nhau), s khng n nh ca on chn v thao tc chng tng i kh khn so vi cc h thng vi khun. Trc nm 1987, cc h thng to dng thch hp ch yu da trn E. coli v ch c th nhn cc on DNA tng i nh. Cc vector vi khun c kh nng ln nht cng ch c th nhn cc on DNA trong phm vi kch thc t 35-45 kb. Tuy nhin, cc YAC vector c th to dng cc on DNA c kch thc t 100-2.000 kb. Sau ny, ngi ta cng pht trin cc vector vi khun mi c kh nng to dng cc on DNA ln hn, nhng cho n nay vn cha th t c kh nng nh cc YAC. Kh nng rt ln ny ca YAC c khai thc xy dng cc bn vt l th h th nht v th hai ca genome ngi. Bng 4.1. So snh mt s c im ca cc vector to dng

2. To dng trong YAC Vector YAC c s dng rng ri nht xy dng cc th vin YAC l pYAC4 (Hnh 4.17). Ct plasmid mch vng di 11 kb bng enzyme hn ch BamHI to thnh mt phn t mch thng c cc trnh t telomere (TEL) c hai u. on nm gia hai v tr cha gen his3 l on stuffer di 1,8 kb c loi b. V tr to dng chn on DNA ngoi lai l EcoRI. y l v tr ct phn t mch thng thnh hai nhnh ca YAC, nhnh phi l mt on di 3,4 kb v nhnh tri di 6 kb cha cc nhn t CEN4 v ARS1. V tr EcoRI nm trong gen sup4. Sn phm ca gen ny l mt tRNA t bin c ch s t bin ochre (nonsense) trong gen ade2. Khi mt on DNA c to dng trong gen sup4 th s c ch b o thi v qu trnh chuyn ha adenine b gin on, dn n tch ly tin cht trao i c mu (phosphoribosyl-aminoimidazole) cho ra cc dng ti t hp mu phn bit.

Hnh 4.17. Vector pYAC4. V tr to dng EcoRI trong gen sup4. trp1 v ura3 l cc marker chn lc nhnh tri v phi ca YAC tng ng. TEL l hai trnh t telomere v CEN4 cung cp chc nng phn tm. Hai marker chn lc ca nm men, trp1 v ura3 cc nhnh tri v phi tng ng, m ha cc enzyme cho php cc t bo nm men mang YAC sn xut tryptophan v uracil iu kin de novo. Dng nm men vt ch S. cerevisiae AB 1380 (MATa+, ura3, trp1, ade2-1, can1-100, lys2-1, his5) khng c kh nng ny, v sinh trng ca nm men ny trong mi trng thiu cc cht chuyn ha nh th cho php chn lc dng tnh i vi cc phn t ti t hp cha c hai nhnh ca YAC. Tai liu tham kho/c thm
1. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA and Struhl K. 2002. Short Protocol in Molecular Biology. Vol 1 and 2. 5th ed. John Wiley & Sons, Inc. USA. 2. Glick BR and Pasternak JJ. 2003. Molecular Biotechnology: Principles and Applications of Recombinant DNA. 3rd ed. ASM Press. USA. 3. Maniatis T, Fritsch EF and Sambrook J. 1989. Molecular Cloning-A Laboratory Manual. Cold Spring Habor Laboratory Press, USA.

4. Primrose SB, Twyman R and Old RW. 2001. Principles of Gene Manipulation. 6th ed. Blackwell Science, Oxford, UK. 5. Rapley R and Walker JM. 1998. Molecular Biomethods Handbook. Humana Press Inc. New Jersey, USA. 6. Singleton P and Sainsbury D. 2001. Dictionary of Microbiology and Molecular Biology. 3rd ed. John Wiley & Sons, Ltd. UK.

Colicin: Mt nhm bt ky cua cac protein c san xut bi cac vi khun khac nhau c ch sinh trng hoc git cht cac vi khun khac. 2 Haemolysin: dung huyt t, cht gy tan mu, cht tiu hng cu. 3 Enzyme sa i (modification enzymes): l cc enzyme tc ng qua li vi DNA sa i cu trc hoc sa cha cc tn thng xy ra vi phn t DNA. 4 Enzyme han ch (restriction enzymes hay restriction endonuclease, RE): xem chng 1. 5 Stringent control: Cn gi l kim sot sao chp cht ch (stringent replication control), m ta s gii han nhn bn cua cac plasmid vi s sao chep ca chromosome vi khun. 6 Relaxed control: Con goi la kim soat sao chep lng lo (relaxed replication control): lin quan n kha nng cua mt s plasmid tip tuc sao chep sau khi vi khun ngng phn chia. 7 Gen lacZ': gen ca E. coli m ha b - galactosidase thch hp cho chn lc th bin np bng khun lc xanh ( b -galactosidase s kt hp vi IPTG v X-gal c b sung trong mi trng nui cy) v trng (on DNA ngoi lai xen vo gia gen lac ZZ lm cho gen ny mt hot tnh v th khng sn xut c b -galactosidase). 8 Chromosome walking: K thut dng lp bn nhim sc th t tp hp cc on ct hn ch chng ln nhau. Bt u t chui DNA bit c th pht hin c cc on ct hn ch khc v bn mt vng c bit c xy dng dn dn. 9 in p cao tnh thm ca mng t bo c tng ln s gip cho DNA ngoi lai d dng i vo bn trong. 10 P1 bacteriophage vector. 11 Nhim sc th nhn to c ngun gc t P1 (P1-derived artificial chromosome).
1

Chng 5

Tao dong genomic DNA cua eukaryote va xy dng th vin genomic DNA
Th vin genomic DNA la mt tp hp cac oan DNA cung ai din cho mt genome nguyn ven (hoc gn nguyn ven) cua ca th ma DNA c bt ngun t o.

Cac oan nay nm trong cac vector t sao chep cho phep chung c duy tri va sinh san cung vi t bao cua c th vi sinh vt, nh vi khun Escherichia coli hoc nm men Saccharomyces cerevisiae. Nhng th vin nh th co th bao quan nh la mt ngun c inh cua cac on DNA ai din cho mt c th c bit. Mt phong thi nghim nay co th thu thp th vin genome t mt phong thi nghim khac hoc t mt ngun thng mai nh la mt cach la chon xy dng th vin ring cua minh. Mt khi thu c, cac th vin c dung chu yu hoc sang loc theo cac phng phap khac nhau nhm phn lp mt (cac) trnh t nucleotide quan tm c bit, hoc xac inh vi tri va th t cua cac trnh t trong genome ma t o th vin c xy dng (physical mapping). Xy dng mt th vin genomic DNA bao gm bn giai oan chinh (Hinh 5.1): Ct DNA, gn DNA vao vector, ong goi cac dong tai t hp trong v protein cua virus lam bc trung gian trc khi xm nhim vao t bao vt chu, va chuyn cu truc o vao t bao vt chu. I. Ct genomic DNA bng cac enzyme hn ch a toan b cac gen cua genome vao mt th vin, trc ht genome phai c ct bi enzyme han ch thanh cac oan DNA co kich thc thich hp tuy thuc vao loai vector nhn chung. Genomic DNA thng c ct bng enzyme han ch co trinh t nhn bit 4 bp, v d nh MboI nhn bit trinh t 5-GATC-3 . Tuy nhin, khng phai d dang thu c mt gen nguyn ven nm trn mt oan DNA. Trong thc t, mt gen thng bi ct thanh nhiu oan DNA do vi tri nhn bit cua enzyme nm ngay trong gen.

Hinh 5.1. Xy dng th vin genomic DNA. Cac giai oan trong xy dng th vin DNA trong vector bacteriophage l . a, b, c v d trnh by s khc nhau (nhng chng ln nhau) ca cc on DNA trong genome, c hp nht trong cc vector ring r v i din nh l cc dng ring bit trong mt th vin. Thng thng, mt phan ng ct tng phn genomic DNA c tin hanh bng cach dung mt lng ti thiu enzyme va u trong mt thi gian ngn sao cho moi vi tri nhn bit khng bi ct ng thi . Do cac vi tri ct c phn b ngu nhin nn mt gen vn co th c bao toan nguyn ven ngay khi no cha vi tri nhn bit trong gen. DNA sau o c phn oan va chi co cac oan co kich thc nm trong pham vi mong mun c chon loc. Cac oan DNA c a vao vector thich hp. Tp hp cac vector cha cac oan genomic DNA tao thanh th vin genomic DNA. Mi oan DNA cha trong vector cua th vin c goi la mt dong (clone). Ty thuc vao kich thc cua cac oan DNA ma chon vector la phage (co th cha oan DNA dai t 15-23 kb), cosmid (45 kb) hay BAC (>100 kb). Co th s dung DNA tach t cac m khac nhau xy dng th vin genomic DNA. Cac th vin genomic DNA cua mt c th chi cha cac oan DNA dai ngn khac nhau nhng thng tin di truyn khng thay i. Ngc lai, th vin cDNA c xy dng t cac mRNA. Trong c th co nhng gen chi hoat ng trong nhng loai t bao

nht inh, do o gia cac m khac nhau co th thu c cac phn t mRNA khac nhau. Vi vy, mt c th co nhiu th vin cDNA khac nhau c trng cho tng loai t chc chuyn hoa (xem chng 6). II. To dng cc on DNA xy dng th vin genome Cac oan DNA thu c t phan ng ct han ch c gn ng ha tri in vitro vao vector tao dong bng cach dung enzyme DNA ligase. Trc khi gn, vector c ct ra mt vi tri n, thng thng la cung vi enzyme c dung ct DNA ngun, nhm tao cac u kt dinh b tr cho cac u cua cac oan chn DNA. Mt i khi, vector va DNA ngun c ct vi t hp hai enzyme khac nhau ngn can mi loai phn t t gn lai. S t tai tao lai vong cua vector cung co th giam thiu bng cach x ly vi enzyme alkaline phosphatase loai nhom 5 phosphate khoi cac u cua vector. Mt s lng ln cac vector tao dong c phat trin cho vic xy dng cac th vin DNA, chon la vector nao phu thuc vao pham vi kich thc cua cac oan DNA c tao dong, va cac ng dung tip theo. 1. Cac plasmid vector Cac plasmid la cc phn t DNA mach vng ong cng ha tri, siu xon v co kich thc vai kilobase (chng 4). Mc du cac plasmid xut hin t nhin, chu yu vi khun, nhng nhng plasmid dung trong xy dng cac th vin DNA thng c thit k co chu inh vi cac cu truc nhn tao. Cac vector tao dong nay cha cac vi tri nhn bit n cho mt enzyme han ch ma tai o cac DNA ngoai lai co th c chn vo (insertion). Cac vi tri cho nhiu enzyme khac nhau co th c tp hp lai trong vng tao dong (multiple cloning sites) hoc vng a ni (polylinker). Cac plasmid mang b sung mt hoc nhiu gen ch th (marker gene), nh cac gen khang khang sinh, cho phep chi co cac t bao cha plasmid sinh trng trn mi trng chon loc, va cac gen cho enzyme nh -galactosidase cho phep cac phn t tai t hp cha oan chn DNA c phn bit vi cac th khng tai t hp. Nhc im cua cac plasmid c dung lam cac vector tao dong la kha nng cha cua chung bi gii han chi thich hp vi nhng oan DNA ngoai lai co kich thc nho vai kilobase (kb) va hiu sut bin nap cua chung vao t bao vt chu tng i thp. 2. Cac bacteriophage vector Cac bacteriophage la cac virus xm nhim vao vi khun. Genome cua chung co th la DNA si i mach vong hoc mach thng, va c boc bng vo protein lam trung gian cho s xm nhp cua chung vao t bao vt chu. Bacteriophage la mt trong cac vector tao dong c ngun gc virus thng dung nht c dung trong xy dng th vin. Genome cua no bao gm mt phn t DNA si i mach thng dai khong 50 kb, phn t nay xm nhim vao E. coli va tao vong bng cach ghep cac u dinh (xem chng 4). Trong chu ky sinh tan (lytic cycle), cac

phn t dng vng cua genome s c sao chp va sau o c ct vi tri c bit (vi tri cos) tao ra cac monomer se ong goi trong cac u protein hoan chinh. Cng c khi phage khng i vo chu ky tan, m chuyn sang giai oan tim tan (lysogenic), trong sut giai oan nay no hp nht n inh trong nhim sc th cua t bao vt ch E. coli . Cac gen oi hoi cho chc nng sinh tan c tp hp lai trong on stuffer, va c loai bo khi xy dng cac vector tao dong co ngun gc co th nhn cac on DNA c kich thc ln n 20 kb. Cac vector tai t hp bao gm nhanh trai, nhanh phai va oan DNA ngoai lai c gn vao gia hai nhanh. Kt qua cac th tai t hp sau o c ong goi trong vo protein bng cach dung h thng ong goi in vitro cho phep xm nhim vao t bao vt chu vi hiu sut cao. i vi genome c trng cua ng vt co vu dai 3 109 bp, xp x khoang 7 5 10 th tai t hp mang cc on chn co kich thc trung binh khong 20 kb s m bao xac sut 99% moi on DNA cn thit hin din trong th vin. 3. Cac cosmid vector Cosmid la plasmid vector mang u cos cua (chng 4). Vi th, cac cosmid co th c ong goi trong cac tiu th vi hiu sut cao. Sau khi xm nhim vo t bao vt chu, DNA tai t hp mach thng tao vong u cos va tip o c nhn ln nh la mt plasmid ln. Ging nh plasmid, cosmid tng i d thao tac, nhng co th nhn cac oan chn genomic DNA co kich thc khoang 40-45 kb, ln hn kh nng ca phage . 4. Cc BAC vector Cc BAC vector hin nay c xem nh cng c hu hiu nht trong vic xy dng cc th vin genome, do chng c cc u im sau: - C kh nng gn cc i phn t DNA 100-300 kb. - t hnh thnh th khm (chimera). - C hiu qu cao trong to dng v thu hi DNA. - Duy tr s n nh ca cc DNA c gn vo vector. Nh nhng u im trn nn h thng BAC thng c dng xy dng bn vt l. DNA c th d dng c phn lp trong cc dng BAC, cc dng phn lp c t lai khun lc s c kim tra bng k thut DNA fingerprinting thng qua phn ng ct ca enzyme hn ch HindIII. K thut fingerprinting cung cp cho chng ta thng tin v nhng phn trng lp (overlapping) v khng trng lp ca BAC. Nh vy, c th hon thin bn vt l c cht lng cao, phc v cho vic phn tch genome v chuyn np gen sau ny. 5. Cc YAC vector

Cc YAC l cc nhim sc th nhn to ca nm men v cng l cc vector to dng c s dng trong phn tch genome. Chng mang cc on telomere (phn cui) v centromere (phn tm) ca mt nhim sc th trong nm men. C hai nhn t ny cn thit cho s ti bn ca nhim sc th trong cc t bo nm men: nu khng c telomere, th sau cc u ca nhim sc th c kh nng b ri ra hoc gn vo cc nhim sc th khc. Nu khng c centromere, th sau cc nhim sc th mi c to thnh s khng c y vo trong cc t bo mi ca qu trnh phn chia t bo. Ngoi ra, cn phi c mt gc ti bn sao chp DNA. Cc nhn t ny c t trong mt on DNA n, c dng nh mt vector to dng DNA ngoi lai trong nm men. u im ca YAC l c th nhn cc on DNA c kch thc ln. Nh vy, trong khi to dng vi khun theo phng php truyn thng bng cch dng bacteriophage hoc cc plasmid thng b gii hn bi kch thc ca cc on DNA c to dng (ch vi chc kilobase), th cc YAC c th to dng cc on DNA di hng ngn kilobase. iu ny gip cho vic lp bn genome hon chnh d dng hn nhiu. Nhc im ca cc YAC l k thut thao tc cn nhiu kh khn, v mt vn chung l DNA t hai vng khc nhau ca genome gc c th kt thc trong mt YAC, dn n xut hin s lin kt sai m kt qu l to ra mt dng khm. III. ng gi in vitro DNA ti t hp v xm nhim vo t bo vt ch Trng hp s dng vector mang gen ngoi lai l bacteriophage hoc cosmid xy dng th vin genome, th th ti t hp trn (naked recombinant) ca cc loi vector ny khng th chuyn vo trong vi khun c. Do , ngi ta cn phi ng gi in vitro DNA ca chng trong mt u rng ca phage , ri sau mi cho chng xm nhim vo cc t bo vi khun. Nguyn l ca s ng gi in vitro (in vitro packaging) l nh vo kh nng mang cc u cos ca phage hai u 5 v 3 ca cc vector ti t hp ng gi chng khi c mt cc u rng ca phage v cc protein ng gi. Phng thc ny i hi cc phn t vector ti t hp phi c chiu di t nht l 38 kb v khng c ln hn 52 kb. Cc u phage c ng gi s hon thin trong cc tiu th phage xm nhim in vitro khi c mt cc sn phm ca cc gen W v FII, v cc ui ca phage. Tt c protein cn thit cho ng gi c th thu c t cc chng E. coli BHB2688 v BHB2690. Cc hn hp ng gi ch cn chun b mt ln v c th bo qun -80oC. Ty thuc vo loi vector m c th dng mt lng thch hp chng E. coli cho vic xm nhim. Cc chng ny c cho i (starve) trc khi s dng, hoc bng cch sinh trng trn mi trng ti thiu v sau x l vi dung dch CaCl 2 0,1 M, hoc bng cch ra trong dung dch CaCl2 0,1 M sau khi nhn ln trong mi trng LB1. Phng thc x l ny cm ng s tng hp cc receptor (protein lamB) trn b mt t bo v tng ng k s hp th phage vo cc t bo E. coli. Cc tiu th

phage xm nhim, thu c khi c mt cc protein phage v cc hp phn ca ui phage, s c dng xm nhim cc vi khun mn cm (susceptible bacteria). Cc dng ti t hp c chn lc trn c s tnh khng khng sinh ca chng v sau c th c khuch i tip. S xm nhim c thc hin bng cch hn hp cc tiu th phage xm nhim thu c sau khi ng gi in vitro cc phn t DNA ti t hp vi cc t bo mn cm (sensitive cell) ca vi khun c tin x l. Tip theo bc ny l khuch i th vin sau khi chun (titration) cc th ti t hp. Th vin gen sau khi c xy dng c th bo qun trong mt vi nm di dng dch huyn ph 4oC, hoc lu hn trong dung dch stock c b sung glycerol (khong 30%) -80oC. IV. Phn tich genomic DNA bng lai Southern Vic phat hin va xac inh cac chui nucleic acid c trng la cng vic thng xuyn trong nghin cu sinh hoc phn t. Nguyn tc cua ky thut nay la da vao s lai phn t (molecular hybridization), di cac iu kin thich hp hai chui nucleic acid n tao thanh mt phn t lai. Phan ng lai phu thuc rt nhiu vao mc tng ng cua hai trnh t nucleotide. Vic tao thanh phn t si i nh th xay ra chu yu thng qua lin kt hydrogen gia cac base G vi C, va A vi T. Thanh phn va s phn b cua cac base khng ging ro rt vi cac phn t nucleic acid cho kt qua cac tinh cht lai khac nhau, vi th lin kt hydrogen (hoc lai phn t gia cac base tng ng) khi c ghep cp thich hp a cung cp mt cng cu co gia tri xac inh cac chui lin quan hoc ng nht vi chui nucleic acid quan tm (mu d). Trong s cac ky thut khac nhau s dung lai phn t phn tich nucleic acid, thi ky thut c s dung ph bin nht la lai mu do nucleic acid c anh du vi nucleic acid ich c c inh trn mt vt rn, thng la mang nitrocellulose hoc nylon. Trinh t cua ky thut lai Southern blot bao gm cac bc sau: (1) phn tch cac oan ct han ch cua genomic DNA bng in di agarose gel, (2) chuyn DNA t agarose 32 gel ln mang lai, (3) lai cac mu do c anh du ng vi phong xa P vi cac nucleic acid c c inh trn mang lai (Hinh 5.2). 1. Phn tch cac oan ct han ch cua genomic DNA bng in di agarose gel DNA mang chui ich (chng han genomic DNA) c ct bng mt hoc mt vai enzyme han ch se cho mt tp hp cac oan co cac chiu dai khac nhau c phn on theo kich thc bng in di trn agarose gel. trng hp genomic DNA hinh anh in di se cho mt vt dai (smear) trn gel (Hnh 5.3). Lng DNA dung cho phan ng ct han ch ty thuc vao mc phong phu cua chui ich (target) co trong mu. Trng hp genomic DNA, ta cn thy phn mt

lng tng i ln DNA (2-10 m g). Nhng nu mu DNA c tao dong trong cac plasmid hoc phage thi chi cn mt lng DNA it hn nhiu (mt vai picogram) la u. Anh gel nhum EtBr c xp thng hang vi thc huynh quang trc khi thm tich la bc quan trong anh gia cac kich thc cua cac bng DNA c lai vi ch th kch thc chun ca DNA (DNA size-marker). DNA cua bacteriophage l c phn ct bng HindIII hoc 1-kb ladder DNA la cac ch th kch thc chun ca DNA thng dung nht cho Southern blot.

Hinh 5.2. S cua ky thut lai Southern blot. Cc mu DNA ch v genomic DNA trong v d ny c ct bng EcoRI v c phn on bng in di trn agarose gel. Cc v tr ct hn ch lin quan v trnh t b sung vi mu d (on dy) cng c trnh by (A v B). DNA ca plasmid mang on chn DNA dng lm mu d (C) c ct v in di lm i chng dng tnh. Sau khi bin tnh v trung ha, DNA si n c chuyn ln mng lai, bng phng thc thm tch mao dn (capillary) hoc bng phng thc thm tch na kh (semi-dry) nh dng in, v c c nh. chun b mu d, on chn DNA (on dy m trong C) c tinh sch t vector bng cch ct v thu hi sau khi in di trn agarose gel, nh du bng 32P v gy bin tnh. Tip theo, mng lai lin kt DNA c lai vi mu d,

tn hiu khng c trng b loi b, mng lai vi phim X-quang. Sau khi ra phim, cc on DNA b sung vi mu d c pht hin nh l cc bng phng x t ghi. Trn hnh ny, nguyn l c bn ca a hnh chiu di cc on ct hn ch c m t. Cc DNA c tch chit t hai mu ring bit (A v B), trong mu B c thm mt v tr EcoRI chui ch. S khc nhau nh th trong genome s c pht hin bi cc kiu lai khc nhau. Nguyn tc ny c khai thc trong nhiu ng dng ca sinh hc phn t.

Hnh 5.3. Hnh nh in di ca genomic DNA sau khi c phn ct bng enzyme hn ch. SM: Ch th kch thc chun ca DNA. PC: i chng dng tnh (on DNA c dng nh du 32P lm mu d). Cc ng 1, 2, 3, 4, 5 v 6: cc mu genomic DNA c ct bng enzyme hn ch. 2. Chuyn DNA t agarose gel ln mang lai Sau khi in di gel va trc khi thm tich, DNA se c kh purin (depurination) bng dung dch HCl loang ct nh DNA tao iu kin d dang chuyn cac oan DNA c kch thc ln ln mang. Nhn chung, bc nay kho kim soat va co th lam mt cac oan DNA nho hn. V th, nu c cach khc phc vic anh gia hinh anh phng x t ghi th c th b qua bc ny. Tuy nhin, kh purin vn cn thit khi phn tich Southern cac oan DNA co kich thc rt ln. Cac loai mang lai c s dung trong Southern la mang nitrocellulose hoc mang nylon. Tuy nhin mang nylon co sc chiu ng cao hn mang nitrocellulose vi th co th tai s dung mt vai ln nn chung c s dung ph bin hn. Hn na, mang nylon co th gn cac oan DNA ngn (<500 bp) hiu qua hn mang nitrocellulose, va DNA sau

khi c thm tich ln mang co th c lin kt ng ha tri bng chiu xa UV trong thi gian ngn (khoang 1-2 phut) 2000 J trong khi o mng nitrocellulose cn un nong 80oC trong iu kin chn khng khoang 2 gi. Mang nylon tich in cung thun li va thich hp cho thm tch kim (alkali-bloting), v d: NaOH, do DNA lin kt cng ha tri vi mang di cac iu kin nay ma khng cn bt ky mt x ly nao na. Trong qua trinh thm tich, thng thng m chuyn c nng mui cao c dung cho genomic DNA, trong khi o DNA ca plasmid hoc cac oan PCR c dung vi cac m c nng mui thp.

Hnh 5.4. S thm tch na kh bng in chuyn DNA t agarose gel ln mng lai

Hnh 5.5. S thm tch mao dn chuyn DNA t agarose gel ln mng lai 3. Lai cac mu do c anh du ng vi phong xa vi cac nucleic acid c c inh trn mang lai Cac iu kin tin lai va lai c thit k tng ti a phan ng lai c hiu cua mu do vi cac chui ich va giam thiu cac lin kt khng c hiu vi mang va DNA khng phai chui ich. Noi chung, cac m lai cn co cng lc ion cao (nhn t quan trong i vi kha nng n inh lai). Mt vai loai tac nhn ngn chn co th c dung c ch cac lin kt khng c hiu, bng cach o a han ch tin hiu nn. Dung dich

Denhardt va sa kh khng c cht bo (nonfat dried milk) c s dung ph bin ngn can (blocking) lin kt gia mu do vi mang lai, cht ty SDS cng c dung trong trng hp ny. Salmon sperm DNA (DNA tinh trng c hi) va calf thymus DNA (DNA tuyn c ca b) c dung ngn can cac tng tac khng c hiu gia cc DNA khng phi chui ch gn trn mang lai vi mu do. Trong gii han thc hanh chi co nhng nhn t anh hng n s n inh nhit cua cac phn t nucleic acid lai mi th hin vai tro quyt inh trong hu ht thi nghim lai trn mang. Nhit nong chay (Tm) cua nucleic acid si i cho bit nhit ma o DNA si i bi bin tinh 50% di cac iu kin a cho, va no la kt qua cua vic o trc tip kha nng n inh cua vic lai nucleic acid. Nhit nng chy cua DNA si i co th c anh gia thng qua biu thc 1:

Trong khi biu thc 2 co th c dung cho th lai RNA-DNA:

Trong o M la nng phn t cua cac cation ha tri 1 (c trng la Na+) (%G + C) la nng phn trm cua guanine va cytosine (%f) la phn trm cua formamide n la chiu dai cua th lai (theo base).

Cng lc dng trong cc th nghim lai l s nghch o ca gi tr chun C (criterion value) c a ra bi biu thc 3:

Trong Ti l nhit th nghim, khi Ti thp th C cao v lc cng lc s thp, v ngc li. Cc chui DNA ch c tng ng ging y ht hoc gn ging vi mu d c th c xc nh di cc iu kin cng lc cao (v d: 5 oC<C <10oC), cc iu kin cng lc thp thch hp cho cc chui t tng ng. Thng thng, phn ng lai v cc bc ra u tin c tin hnh iu kin cng lc thp (v d: Tm = 30oC) v cng lc c tng ln dn trong cc bc ra tip theo. Biu thc 1 v 2 ch chnh xc trong trng hp cc DNA si i di hn 100200 nucleotide. Cc mu d oligonucleotide c dng thng xuyn sng lc cc th vin cDNA hoc genomic DNA v phn tch Northern, v i khi chng cng c

dng trong cc th nghim lai Southern. Cc phn t lai ngn c n nh thp (ngay c khi tng ng 100%), c bit trong sut cc bc ra, bi v nng mu d trong m ra hu nh l bng khng. Nh vy, cc bc ra cn tin hnh trong thi gian ngn (2-5 pht). Hn na, n nh ca cc si i oligonucleotide b nh hng ln bi thnh phn nucleotide v bi hin tng ghp i khng tng xng. Tm ca cc mu d oligonucleotide ngn hn 50 nucleotide thng c tnh bng biu thc 4: Tm (oC) = 4 (G + C) + 2(A + T) (4) Trong A, C, G v T l s cc nucleotide tng ng. Tuy nhin, thng thng cc iu kin th nghim ca phn ng lai ch thc vi cc mu d oligonucleotide (c bit khi phn tch cc genome ln) phi c xc nh trn c s kinh nghim. Cc th nghim lai trn mng thng c tin hnh bng cch nh du mu d bng 32P (mc d cc ng v phng x khc nh 35S cng c th c s dng). Phn ng lai ca Southern i hi hot tnh c hiu ca mu d ti thiu phi l 109 dpm/ g, mc d hot tnh 108 dpm/ g c th c xem nh l chp nhn c trong cc ng dng cn cng lc thp. Cc phng php khng dng ng v phng x (v d: digoxigenin-dUTP) ngy cng c s dng ph bin hn mc d nhy ca chng l khng th so vi cc phng thc dng ng v phng x (Hnh 5.6). Nhng cc k thut khng dng ng v phng x an ton hn cho nghin cu vin, to ra cc mu d c th c bo qun trong thi gian di trc khi dng v kt qu pht hin cc tn hiu lai nhanh hn.

Hnh 5.6. Hnh nh phn tch Southern blot ca hai th nghim khc nhau. (A) Hnh nh phng x t ghi trn phim X-quang vi mu d c nh du 32P. (B) Hnh nh bt mu trn mng lai Hybond-N vi mu d c nh du digoxigenindUTP. Cc bng mu en l tn hiu lai gia DNA ch v mu d. V. Sng lc th vin genomic DNA

DNA c to dng trong cc vector c ngun gc plasmid hoc cc vector nhim sc th nhn to (BAC, YAC) sn xut ra cc khun lc (vi khun hoc nm men) khi cc nui cy bin np c dn mng trn a agar cha mi trng sinh trng v nui di nhng iu kin thch hp. Trong khi , cc vector c ngun gc virus (bacteriophage) s sinh tan t bo b chng xm nhim v sn xut ra cc plaque (vt tan) c dng hnh trn chu vi khong 2-3 mm c mu sng trn thm vi khun (bacterial lawn) ca a agar (Hnh 5.7).

Hnh 5.7. Cc vt tan ca phage l trn thm vi khun E. coli. Cc phage l c trn vi cc t bo E. coli trong mi trng nui ri dn mng (plating) ln a petri cha bottom agar (nui cy top agar). Sau khi qua m 37 oC, cc t bo vi khun mc to nn thm vi khun, trn cc vng b nhim phage l hnh thnh nn cc vt trong, do hin tng sinh tan vi khun ca phage, gi l vt tan. Cc vector, nh m t trn, thng cha cc gen ch th cho php chn lc nhng t bo vt ch mang vector (th bin np). Thng thng cc ch th ny l gen khng khng sinh v cc t bo bin np sinh trng trn mi trng cha khng sinh tng ng. Ngoi ra, cc vector cn cha cc gen b sung phn bit cc t bo bin np cha on chn ca DNA ngoi lai vi cc t bo cha cc vector t ti to li vng. V d: vector m ha gen -galactosidase xc tc sn sinh ra cc sn phm mu xanh t c cht khng mu X-gal cho kt qu sinh trng ca cc khun lc mu xanh. on chn ca DNA ngoi lai lm mt hot tnh ca gen cho kt qu sn xut ra cc khun lc mu trng. Mt dng cha cc chui DNA quan tm c bit c th c xc nh bi lai khun lc. Mt lng nh khun lc bin np hoc vt tan c chuyn ln mng nitrocellulose hoc nylon c ph ln (overlay) trn a agar trc . DNA c bin tnh v c nh trn mng bng cch un nng (baking) hoc chiu tia cc tm (UV-crosslinking), v sau c lai trong m cha mu d c nh du ng v

phng x c trnh t b sung mt phn ca chui c xc nh. V d: C th mt oligonucleotide tng hp nhn to, c ngun gc t genomic DNA tng phn, cDNA hoc chui protein hoc sn phm PCR. Mt vi trng hp mu d c th c thit k da trn trnh t bt ngun t gen tng ng ca cc loi khc v thng c lai vi cng lc thp. Cc mu d tha c ra khi mng vi phim Xquang. Theo hng ca phim (sau khi ra) vi s i chiu a agar gc, sau s c kh nng gn kt cc khun lc thc t vi cc khun lc lai dng tnh tng ng trn phim X-quang (Hnh 5.8).

Hnh 5.8. Sng lc (screening) th vin gen trong khun lc vi khun hoc vt tan ca bacteriophage l Gn y, mt phng php sng lc khc c thit k. Theo hng ny cc khun lc vi khun hoc nm men c nht ring r v chm ngay ngn thnh hng ln mng hoc trong ging ca a microtiter. Mi dng c th c xc nh bng ta ng k ring r ca n. So vi cc phng php truyn thng, phng php mi ny d dng hn trong vic t ng ha, sp xp cc th vin v i chiu s liu gia cc phng th nghim khc nhau. Nu dng mong mun biu hin mt protein c bit hoc mt on protein, th sau bng cch xy dng th vin vi mt vector biu hin cha cc tn hiu phin m v dch m ngi ta c th sng lc trc tip i vi protein c biu hin. V d: nu protein tng ng y l thch hp, th khng th c hiu c th c to ra, c nh du v s dng pht hin yu t quyt nh khng nguyn trn protein c biu hin t cc khun lc c dung ly. Nu on chn c yu cu m ha mt hot tnh sinh hc, th sau vic sng lc c th c thc hin bng cc th nghim cho hot tnh , hoc bng cch chn lc trc tip, v d: chn lc

trn mi trng sinh trng khng hon chnh i vi enzyme sinh tng hp cn thit cho s sinh trng. Tuy nhin, cc hng da trn s biu hin ni chung d ng dng cho th vin cDNA hn l th vin genomic DNA. VI. Cc phng php nh du on DNA bng phng x Mc ch ca bc ny l sn xut cc on DNA c phng x v hot tnh c hiu cao lm mu d dng trong cc th nghim lai phn t. Trong trng hp ny du phng x thng c s dng l 32P pht x b nng lng cao. Di y l mt s phng php nh du ph bin c trong k thut gen. 1. nh du ui Enzyme polynucleotide kinase xc tc chuyn nhm phosphate nm cui ATP sang gc 5-OH ca cc phn t nucleic acid c dephosphoryl ha. Nu nh ATP c nh du phng x, th n s to ra nucleic acid c nh du phng x. Tuy nhin, hot tnh c hiu ca chng tng i thp, do ch c phn ui ca mi phn t DNA c nh du (Hnh 5.9).

Hnh 5.9. nh du ui ca on DNA nh enzyme polynucleotide kinase (PNK). (a) DNA c dephosphoryl ha bng enzyme phosphatase to ra cc nhm 5-OH. (b) Tip , ui phosphate ca [ g -32P]ATP (vng trn m trn hnh) c chuyn sang gc 5-OH nh PNK. y l phn ng trao i cc nhm 5-PO4. 2. nh du bng dch chuyn im t Phng php ny da vo enzyme DNA polymerase I ca E. coli c kh nng dich chuyn im t cua DNA (xem chng 1). Cc im t c th xut hin t nhin v cng c th do tc dng ca enzyme DNase I2 nng thp trong hn hp phn ng. Enzyme DNA polymerase I xc tc phn ng thay th si bng cch xen cc dNTP mi vo chui DNA. Nu mt trong cc dNTP nh du phng x (v d: [ a -32P]dCTP) , th kt qu l phn t DNA s c nh du vi hot tnh c hiu cao (Hnh 5.10).

3. nh du bng ko di on mi on DNA cn nh du c bin tnh bng nhit, sau cc on mi oligonucleotide (thng l cc phn t hexadeoxyribonucleotide) c gn vo cc DNA si n. S dng enzyme DNA polymerase I c th tng hp nn bn sao mi ca si khun mu. Nu nh mt dNTP nh du phng x (v d: [ a - 32P]dCTP) c gn vo, th bn sao DNA mang hot hat tnh c hiu rt cao s c to ra (Hnh 5.11). Cng c th dng k thut PCR nh du phng x on DNA trong trng hp mun to ra mt s lng ln mu d. Lc ny, enzyme DNA polymerase I s c thay bng DNA Taq polymerase.

Hnh 5.10. nh du DNA bng dch chuyn im t. (a) Dng DNase I to ra mt im t trn si n ca chui DNA si i. (b) Tip enzyme DNA polymerase I tng hp mt bn sao mi ca si khun mu khi phn hy si c im t nh hot tnh exonuclease 5 - 3 ca n. Nu [ a - 32P]dCTP c cung cp th n s gn vo bn sao mi (cc hnh trn bi en). Trong phn ng nh du phng x, thng thng cn tch DNA c nh du khi cc nucleotide khng c nh du cn tha trong hn hp phn ng. Hot tnh phng x ca mu d cn c nh gi bng phng php m nhp nhy lng (liquid scintillation). Cc s liu sau c dng tnh ton lng mu d cn thit cho cc phn ng lai phn t (v d: Southern blot, Northern blot, screening).

Hnh 5.11. nh du DNA bng cch ko di on mi. (a) DNA c bin tnh to ra cc phn t si n. (b) Mt on mi oligonucleotide c b sung gn vi si DNA khun mu. (c) Enzyme DNA polymerase I xc tc tng hp mt bn sao mi ca si khun mu bng cch ko di on mi, trong qu trnh cc [ a -32P]dCTP (cc vng bi en) s c nh vo bn sao cc v tr c G. VII. ng dng ca th vin genomic DNA Th vin genomic DNA c cc ng dng trong phm vi rng lp bn vt l ca DNA v xc nh cc gen gy bnh hoc cc chui DNA quan tm cho nhng phn tch xa hn na. Sn xut ra cc dng mang cc on chn DNA khc nhau nhng gi chng ln nhau c nhiu thun li v th vin c th c s dng trong qu trnh chromosome walking. Phng thc ny cho php xc tin t im khi u trn nhim sc th (v d: gen ch th lin kt vi bnh) ti locus khng xc nh gn (v d: gen gy ra chnh bnh ) bng cch tin hnh cc chu k lp li ca to dng, m t c im v lai phn t bng cch dng cc dng nh l mu d xc nh cc dng xa hn vi th vin mang chui gn k t DNA gc. Chromosome walking thng c thc hin vi th vin ca cosmid, l hoc YAC. Chromosome walking cng cho php xy dng mt cu trc clone contig (mt chui tun t cc dng DNA gi chng ln nhau cng vi s hin din ca vng lin k ca genomic DNA). Cu trc clone contig thng to thnh mt phn cn thit ca vic phn tch cc chui DNA c to ra trong sut qu trnh xy dng bn vt l ca DNA v xc nh cc gen gy bnh bng to dng v tr (positional cloning). Cu trc clone contig c xy dng bng cc vector khc nhau, bao gm l , BAC, YAC Xc nh gen gy x nang minh ha cho vic s dng th vin genomic DNA xc nh bng cch to dng v tr. Cc th vin genomic DNA cng hu ch trong vic xc nh cc gen gy bnh bng cch to dng chc nng (functional cloning). Theo hng ny, thng tin v chc nng ca gen c khai thc phn lp gen mong

mun t th vin. Mt oligonucleotide c trnh t da trn chui amino acid tng phn c dng nh l mt mu d phn lp dng cDNA bng cch sng lc th vin cDNA. Dng cDNA ny sau c th c dng trong sng lc th vin genome phn lp cc dng genomic DNA v cho php quan st c im ca chui genome hon chnh. Hng ny c dng xc nh gen hemophilia A (nhn t VIII). Cc mu d oligonucleotide da trn chui amino acid ca protein nhn t VIII ca ln c s dng trc phn lp dng t genome (nhn t VIII ca ln), sau n c dng nh l mt mu d cho th vin DNA ngi xc nh gen ngi. VII. Phn tch trnh t ca on DNA c to dng T cui nhng nm 1970, trnh t cc nucleotide trn phn t DNA c xc nh mt cch n gin v nhanh chng nh s ra i ca hai phng php khc nhau: phng php ha hc v phng php enzyme. Tuy nhin, t nhng nm cui ca th k 20, trnh t nucleotide c xc nh trn my c t ng nh tr gip ca computer ngy cng ph bin. u im ca phng php ny l gim cc thao tc, tit kim ha cht v trnh t c c di hn hn so vi cc phng php trc . 1. Phng php ha hc Maxam-Gilbert u tin phn t DNA si i c nh du 32P ti u 5. Sau , hai si n tch ri nhau do bin tnh nhit. Chng c x l vi cc cht ha hc sao cho ch mt loi nucleotide b ph hy. Trn hnh 5.12 l nucleotide A. Nng cc cht ha hc c kim tra cht ch sao cho ch mt nucleotide b ph hy trn mi si n DNA. Kt qu hng lot cc on DNA c kch thc khc nhau c to thnh. Cc on DNA to ra do b b gy c phn ly trn polyacrylamide gel v ch c on no b gn 32P u 5 mi quan st c. Kch thc ca chng tng ng vi khong cch t u 5 c nh du n v tr ca cc nucleotide b b gy trn phn t DNA. S dng cc cht ha hc khc nhau ph hy bn loi nucleotide bng bn phn ng ring bit. Sn phm thu c ca bn phn ng ny c phn ly trn cng mt gel. on DNA ngn nht c nh du phng x chy nhanh nht di tc dng ca in trng. Cn c vo v tr cc vch trn gel m xc nh trnh t cc nucleotide (Hnh 5.12).

Hnh 5.12. Xc nh trnh t nucleotide bng phng php ha hc. A: tin hnh bn phn ng c lp, s dng bn loi ha cht khc nhau, mi ha cht ch ph hy mt loi nucleotide nht nh (trong hnh v l A). B: Sn phm ca bn phn ng c chy in di ng thi trn polyacrylamide gel. Trnh t nucleotide c t di ln theo chiu 5 - 3. 2. Phng php enzyme Sanger y l phng php to u tn cng ca chui (chain terminator method). Nguyn l ca phn ng ny l s dng dideoxynucleotide khng c nhm OH v tr 3 trong phn ng tng hp DNA. Do , khi DNA polymerase gn chng vo si DNA th qu trnh tng hp b ngng li. V vy, phng php ny cn gi l phng php dideoxy (dideoxy method). u tin si i DNA (si khun cn c trnh t) b bin tnh tch thnh hai si n v c lai vi primer. Primer l mt si n gm vi chc nucleotide c th lin kt vi mt trong hai si n DNA theo nguyn tc to cp b sung. Sau , bn phn ng ring r c tin hnh ng thi. Mi phn ng l hn hp ca DNA khun mu, primer, DNA polymerase, mt loi dideoxynucleotide v bn loi deoxynucleotide theo t l 1:100. Cc si n DNA c tng hp c di khc nhau do dideoxynucleotide gn ngu nhin lm dng phn ng. Sn phm ca bn phn ng cng c phn ly trn polyacrylamide gel cho php phn bit hai si n DNA hn km nhau mt nucleotide. Cc vch DNA quan st c nh c gn phng x 32P vo mi hoc vo mt trong bn loi loi nucleotide bnh thng (Hnh 5.13).

3. Xc nh trnh t nucleotide trn my t ng Trong nhng nm gn y, mt s phng php xc nh trnh t mi xut hin. Bn cnh k thut thng thng s dng cc gel phn ly cc phn t DNA c di khc nhau, cc k thut mi lin quan n pht hin hunh quang ca cc nucleotide c nh du, phn tch trnh t DNA bng khi ph, in di mao qun hoc lai vi cc on oligonucleotide c tng hp nhn to. Nhng tin b nhanh chng trong lnh vc knh hin vi in t cho php quan st chi tit cu trc b mt ca phn t nucleic acid v phn bit tng nucleotide. Ngoi ra, k thut lai s dng tm chip gn c nh cc oligonucleotide cho php phn bit c hn 50.000 phn t DNA khc nhau. Trong tng lai gn, k thut lai ny c th gn hng nghn oligo trn mt tm chip nh v trnh t nucleotide c phn tch t ng bng cc chng trnh ca computer. iu cho php xc nh trnh t mt cch nhanh chng.

Hnh 5.13. Xc nh trnh t nucleotide bng phng php enzyme (Sanger). Dideoxyribonucleotide triphosphate (ddNTP) gn vo si DNA ang tng hp s lm ngng phn ng. Nh thu c cc on DNA hn km nhau ch mt nucleotide. Cc on ny c quan st trn polyacrylamide gel khi primer c nh du phng x 32P hoc nh du hunh quang. Phng php to u tn cng ca chui c ci tin cho php xc nh trnh t trn my c t ng (automatic sequencer), s dng cc b Kit khc nhau, trong u tn cng c nh du bng cc cht pht hunh quang mu (dye terminator). Phn ng gn cc nucleotide nh du vo si DNA tng hp trn khun mu c tin hnh trong my PCR. V vy, k thut ny cn c gi l phn ng chui c trnh t. Sau , sn phm PCR c in di trn polyacrylamide gel c phn gii cao, cho php phn bit c cc si n DNA hn km nhau mt nucleotide. Qu trnh chy in di c thc hin trn my t ng v kt qu c phn tch bng cc chng trnh computer chuyn dng (Hnh 5.14). Trn cc my c t ng hin i, thng thng bn loi nucleotide c nh du bng cc cht pht hunh quang khc nhau. u c laser trong my s kch hot cc cht ny khin chng hp th v pht ra cc mu, mi mu ng vi mt loi nucleotide v c hin th bng mt nh. Thng thng, phn ng gn nucleotide vo si DNA tng hp trn si khun mu c thc hin bng PCR. Hai loi nucleotide dGTP v dTTP c thay th bng dITP v dUTP nhm hn ch s trng lp cc nh. Sn phm PCR thng c tinh sch, loi b cc nucleotide v primer tha trc khi a vo my c t ng. Trnh t c c trn my t ng thng di khong 600-1.000 bp. u im ca phng php ny l kt qu c c a trc tip vo chng trnh computer, gim bt sai st do c bng mt gy ra. Hn na, phn ng PCR khng i hi lng DNA khun mu (dng si i) nhiu nhng cc si n DNA cn c li c khuch i ln rt nhiu ln.

Hnh 5.14. My phn tch trnh t nucleotide t ng v hnh nh in di cc bng DNA pht hunh quang quan st c trn computer Trnh t h gen cng nh cc loi DNA c xc nh ngy cng nhiu cc sinh vt khc nhau. Do , vic lu tr s liu, phn tch, so snh v sp xp chng thc y hnh thnh mt hng nghin cu mi l tin sinh hc (bioinformatics). Mi lin quan mt thit gia trnh t nucleotide v protein khin lnh vc mi ny pht trin rt nhanh chng. Ba ngn hng d liu chnh hin nay lu tr hu ht cc thng

tin v DNA l EMBL (thuc European Informatics Institute), GenBank (thuc US National Centre for Biotechnology Information) v DDBJ (thuc DNA Database Bank of Japan). Mt ngn hng d liu khc lu tr thng tin v protein l Swiss Protein Database (Thy S).

Hnh 5.15. Minh ha trnh t nucleotide mt on DNA c phn tch trnh t

Tai liu tham kho/c thm

1. V Th Thng Lan. 2002. Sinh hc phn t. NXB i hc Quc gia H Ni, H Ni. 2. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA and Struhl K. 2002. Short Protocol in Molecular Biology. Vol 1 and 2. 5th ed. John Wiley & Sons, Inc. USA. 3. Brown TA. 2001. Gene Cloning-An Introduction. 4th ed. Blackwell Science, Oxford, UK. 4. Glick BR and Pasternak JJ. 2003. Molecular Biotechnology: Principles and Applications of Recombinant DNA. 3rd ed. ASM Press, USA. 5. Maniatis T, Fritsch EF and Sambrook J. 1989. Molecular Cloning-A Laboratory Manual. Cold Spring Habor Laboratory Press, USA.

6. Ohman DE. 1989. Experiments in Gene Manipulation. Prentice Hall, Englewood Cliffs, New Jersey, USA. 7. Primrose SB, Twyman R and Old RW. 2001. Principles of Gene Manipulation. 6th ed. Blackwell Science, Oxford, UK. 8. Rapley R and Walker JM. 1998. Molecular Biomethods Handbook. Humana Press Inc. New Jersey, USA. 9. Surzycki S. 2000. Basic Techniques in Molecular Biology. Springer-Verlag, Berlin, Heidelberg, Germany. LB: lysogeny broth, mt mi trng giu dinh dng c dng ch yu cho s sinh trng ca vi khun. Mi trng LB cn c gi l Luria broth hoc Luria-Bertani broth.
1 2

DNase I (ty ca b): enzyme xc tc phn ng thy phn lin kt nm ngay sau mt pyrimidine trn chui DNA. Trong trng hp DNA si i, ch ct c th xy ra trn mt hay trn c hay si (xem chng 1).

Chng 6

Tao dong va xy dng th vin cDNA

I. Tach chit, tinh sach v phn tch RNA 1. Tch chit v tinh sch RNA 1.1. Tach chit RNA tng s - Kim soat hoat tinh ribonuclease. Cac phn t RNA khng bn, d bi phn hy bi cac ribonuclease (RNase). thu c dich chit RNA c cht lng tt, cn phi giam thiu hoat tinh cua RNase c giai phong trong sut qua trinh sinh tan t bao hoc t cac ngun tim tang khac trong phong thi nghim (dung cu, ban lam vic, ban tay cua ky thut vin...) bng cac nhn t c ch RNase, bao gm cac nhn t c ch protein cua RNase, cac phc hp vanadyl-ribonucleoside hoc macaloid. - Nguyn ly. Phng phap tach chit RNA tng s bao gm cac bc c ban ging nh tch chit DNA. T bao hoc m c nghin trong dung dich co cha cht ty manh la SDS (sodium dodecyl sulphate, sarcocyl) nng cao, cac dung dich mui manh (guanidinium thiocyanate-GTC) ha tan RNA va kt tua cac protein bi bin tinh cung mt thi gian, v mt cht kh (-mercaptoethanol). Hai loai cht sau co tac dung c ch cac RNase ni bao va tach cac protein lin kt khoi phn t RNA. Bn canh o, co th b sung cac protease (chng han protease K) gy bin tinh cac thanh phn protein cua phc cht RNA-protein. Cac protein c loai bo khoi mu bng cch chit

(ly tm) vi phenol, phenol:chloroform va chloroform. RNA ha tan trong pha nc c kt tua bng ethanol (hoc isopropanol) va c thu nhn lai qua ly tm. RNA o c bao quan trn mt nm -70 C trong nc co cha RNasin (nhn t c ch RNase). 1.2. Phn lp RNA poly(A)+ RNA tng s cua t bao cha khoang 90-99% rRNA va tRNA, trong o rRNA chim khoang 80-85% va tRNA chim khoang 15-20%. Cac RNA nay co kich thc va trinh t xac inh va co th c tach ring d dang bng in di hoc ly tm. Ring mRNA chi chim khoang 1-5% RNA tng s cua t bao, lai co kich thc va trinh t rt a dang. Tuy nhin, chung co mt im chung la cu truc ui polyA (co th ln ti 100A). Da vao cu truc nay va c tinh lin kt b sung A-T cua nucleic acid, co th tach mRNA ra khoi mu bng sc ky ai lc trn ct oligo(dT)-cellulose. Cac mRNA se bam ln ct thng qua lin kt b sung vi oligo(dT) va c thu nhn lai qua ly tm. Ky thut nay cho phep thu nhn mRNA t nhng mu co khi lng rt nho.

Hnh 6.1. S quy trnh tinh sch mRNA t RNA tng s

2. Phn tch RNA 2.1. Lai Northern (Northern hybridization) Phn tich RNA la cng vic quan trong cua nhiu nghin cu v sinh hoc phn t, vi no co th cung cp thng tin v s biu hin cua gen trong c th sng. Lai bng mang loc (nylon hoc nitrocellulose) cac RNA c phn oan vi oan mi la nucleic acid 32 anh du ng vi phong xa P (hoc digoxigenin-dUTP) c goi la phng php thm tich Northern (Northern blot). Phng thc nay bt ngun t phn tich Southern blot (xem chng 5), da trn c s kha nng b sung cua cac nucleic acid si n tao thanh cac phn t lai. Mt cach tom tt, RNA c phn chia theo kich thc trn agarose gel di cac iu kin bin tinh, thm tich va lin kt khng thun nghich vi

mang nylon hoc nitrocellulose, lai vi oan mi nucleic acid c anh du 32P. Sau khi ra tri oan mi khng lin kt hoc lin kt khng c hiu, cac phn t RNA lai c phat hin nh la cac bng phong xa t ghi trn phim X-quang (Hnh 6.2).

Hnh 6.2. Phn tch s biu hin ca gen bng lai Northern. Hnh pha di l in di RNA tng s trn agarose gel c bin tnh bng formamide. Hnh pha trn l tn hiu phng x thu c t phn ng lai gia RNA tng s vi mu d c nh du bng 32P.

Thm tich Northern la phng php quan trong nghin cu biu hin cua gen, do cac RNA hin din trong t bao hoc loai m quy inh m ta khai qut mt phn cua genome c biu hin trong t bao hoc m o. Phn tch Northern cho thy mc n inh cua s tich ly chui RNA qui inh (thng la mRNA) trong mu nghin cu. Vi th, phn tich Northern cho phep ngi ta lin kt cac mc biu hin mRNA vi cac tinh cht hinh thai va sinh ly cua c th sng. Ky thut lai Northern l mt phng php c ng dung rng rai cho vic phn tich biu hin gen. Vi du: ky thut nay a c s dung cho vic m ta c im cua cac cDNA va gen c tao dong, nghin cu c trng c quan/m cua s biu hin gen, phn tich hoat tinh cua cac gen ni va ngoai sinh trong c th chuyn gen, va nghin cu s biu hin cua gen trong mi lin quan vi s phat trin va cac yu t v sinh va hu sinh.

2.2. Lai Dot (Dot hybridization) Lai dot c Kafatos v cs. (1979) thc hin u tin bng cch chm cc mu nh ca dung dch RNA ln mng nitrocellulose, sau mng c lm kh, lai vi mu d c trng RNA hoc DNA c nh du 32P, v vi phim X-quang. Mc d kh nh lng chnh xc do kch thc ca cc chm ln v khc nhau, nhng trong nhiu trng hp c th thu c mt tng tt v cng biu hin ca mt gen c bit no trong cc m c trng hoc cc t bo nui cy (Hnh 6.3).

Hnh 6.3. Phn tch dot nh lng RNA. Hng pha di l cc nng RNA chun bit. Hng trn l cc nng RNA khc nhau c tch chit t m/c quan.

II. Tng hp cDNA (complementary DNA) Phng phap tng hp cDNA co nhiu thun li do: (1) Ly mRNA ung cua gen. (2) Khng tn cng phn tich nhng oan gen trung lp. (3) Chon loc d dang trong mt s trng hp. y chi trinh bay phng phap tng hp cDNA. Qua trinh tng hp cDNA qua ba giai oan nh sau: (1) Tng hp si th nht bng enzyme reverse transcriptase. (2) Bin tnh v ct RNA trong th lai mRNA-cDNA tun t bng nhit v enzyme RNase H ca E. coli. Thay th khun mu l chui RNA bng chui cDNA th nht va tng hp si cDNA th hai nh DNA polymerase I ca E. coli. (3) Ct vng cp tc cua cDNA si i nh nuclease S1 v sa cha hai u bng on Klenow (Hnh 6.4).

1. Tng h p s i cDNA th nht 1.1. Enzyme phin m ng c (reverse transcriptase) Nhn t quan trong nht trong tng h p cDNA la cht l ng cua enzyme phin m ng c c s dung trong phan ng. Mc du cht l ng cua enzyme c s dung noi chung la tt, song vn kho loai tr c RNase bn. Tr ngai nay co th khc phuc bng cach dung cac nhn t c ch manh RNase (RNase inhibitor), vi du nh: vanadyl-ribonucleoside hoc RNasin, trong phan ng phin m ng c. Ty l enzyme phin m ng c dung cho khun mu mRNA cung co anh h ng khac nhau n hiu sut tng h p cDNA hon chinh. Nu s l ng khun mu nhiu thi hiu sut cua san phm phin ma cDNA se tng ln cung v i vic tng s l ng enzyme reverse transcriptase. Trong nghin c u, hiu sut ti a cua cac san phm phin ma cDNA hon chinh at n 80 tiu phn enzyme/ g cua khun mu, ty l enzyme cao nh th oi hoi phai s dung enzyme tinh sach cao va bao gm ca cac nhn t c ch RNase trong phan ng.

1.2. Oligo dT primer Cac oligo dT primer c s dung lam mi cho phan ng tng h p s i cDNA th nht d a trn khun mu cua mRNA. Cac oligo dT primer se lin kt v i ui polyA cua mRNA.

1.3. Deoxynucleoside triphosphate (dNTP) Nng cua bn loai dNTP la yu t c bit quan trong anh h ng n hiu sut tng h p cDNA. Nu nng cua mt trong bn dNTP giam xung d i 10-50 M thi hiu sut cua san phm phin ma giam ro rt. Khi s dung RNA cua avian myeloblastosis virus lam khun mu thi vic san xut cac cDNA hon chinh tng ti a nng 75 M cua ca bn loi dNTP. Nng cua dNTP t 200-250 M ang c dung ph bin.

1.4. Cac cation hoa tri 1 va hoa tri 2 Cac iu kin ion anh h ng tht s n hiu qua phin ma cua cac khun mu + khac nhau. i v i cac san phm phin ma dai thi ion K co hiu qua cao hn ion + + Na . Nng K ti u cho qua trinh tng h p va keo dai kich th c cDNA la khoang t 140-150 mM. Cac cation ha tri 2 la yu cu tuyt i cho hoat tinh cua enzyme phin ma ng c. Khng co hoat tinh nao c tim thy nng <4 mM 2+ 2+ cua ion Mg , nng ion Mg ti u cho qua trinh san xut cac san phm phin ma hon chinh la khoang 6-10 mM.

1.5. pH cua dung dich phan ng Gi tr pH 8,3 la ti u san xut hiu qua cac san phm phin ma cDNA hon chinh. Trong qua trinh san xut cac san phm phin ma, mt s m a c th nghim nhng khng tt hn m Tris.HCl.

2. Tng hp si cDNA th hai Gy bin tinh (un si) th lai mRNA-cDNA ct RNA bng RNase H ca E. coli. Thng thng, u tn cung 3 cua cac cDNA si n co kha nng tao thanh cac 1 cu truc vng cp toc (hairpin loop) va vi th co th c s dung lam mi (primer)

cho qua trinh tng hp si cDNA th hai bng DNA polymerase I cua E. coli hoc reverse transcriptase (Hinh 6.4). Mc du ngi ta a d oan cu truc vong i u tn cung cua cac cDNA va c ch phat sinh cua chung, nhng hin tng tng hp si cDNA th hai vn cha c nghin cu mt cach h thng. Tng hp si cDNA th hai bng DNA polymerase I a c s dung rng rai. oan Klenow cua DNA polymerase I thiu hoat tinh exonuclease 5' cung c s 3' dung thanh cng tng hp si cDNA th hai. Nhiu tac gia a s dung reverse transcriptase tng hp si cDNA th hai. Mc du co tac gia cho rng AMV reverse transcriptase khng th dung tng hp si th hai cua cDNA immunoglobulin, nhng thanh cng cua nhiu thi nghim dung reverse transcriptase tng hp si cDNA th hai a cho thy vic s dung ca hai enzyme u co th cho kt qu tt. DNA polymerase I va reverse transcriptase co th tam ngng hoc ngng lai cac chui khac nhau. Vi vy, cac si th hai c tng hp mt cach c bit, chung c san xut nh mt enzyme va c m rng hon toan nh mt enzyme khac.

Hinh 6.4. S tng hp on cDNA t khun mu mRNA

3. Ct vong cp toc nh nuclease S1 Sau khi tng hp cDNA hon toan, si th nht va th hai c lin kt cng hoa tri bi vng cp toc va vong cp toc d bi ct bi nuclease S1. Sau , on cDNA c sa cha bng enzyme Klenow, kt qua la hai u tn cung la u bng. Si i cDNA sau o c tach thanh cac tiu phn theo kich thc va cac phn t ln nht c gn vao cac plasmid cua vi khun. Hoc la mt tp hp y u cac kich thc cua cDNA si i c tao dong trong bacteriophage xy dng th vin cDNA (cDNA library). Tuy nhin, vic a cac u bng vao vector s gy kho khn trong vic ly chung ra khoi vector mt cach nguyn ven sau ny. Do o cac linker thng c ni vao hai u cua cac cDNA nh DNA ligase. Linker la nhng oan nucleotide ngn co cha vi tri nhn bit cua mt loai RE (vi du: EcoRI) c tng hp nhn tao tng ng vi vi tri nhn bit RE (vi du: EcoRI) cua vector. Sau o, cac cDNA mang linker va vector se c ct bi cung mt enzyme (vi du: EcoRI). Nh o cac cDNA va vector u co u sole tng ng (u dnh) va cDNA se d dang gn cung nh ly ra khoi vector mt cach nguyn ven.

III. Tao dong phn t cua cDNA si i Co nhiu phng phap khac nhau c dung lin kt cDNA si i vi cac plasmid vector. a s c dung theo cc phng php sau: - B sung cac ui ng trung hp (homopolymetric tailing) cho cDNA si i va cho DNA ca plasmid vector. DNA vector va cDNA sau o c ni bng lin kt hydrogen gia cac ui ng trung hp b sung. S tao thanh vong DNA ong bng enzyme gn in vitro (DNA ligase) la cn thit hinh thanh nn cac plasmid tai t hp trong E. coli. - B sung cac on ni nhn tao (synthetic linkers) cho u tn cung cua DNA si i. Sau khi phn ct bng RE thich hp, cac phn t DNA c chuyn vao trong plasmid DNA cung a c ct vi cng mt enzyme.

1. ui ng trung hp 1.1. ui dA:dT Enzyme terminal transferase xuc tac cho s b sung cua cac deoxyrinucleotide triphosphate (dNTP) vao u 3-OH cua DNA si i hoc si n, c dung a DNA tai t hp vao trong E. coli bng cch ni dA:dT. Thng thng t 50-150 gc dA c b sung vao vector DNA va mt s tng ng cua cac gc dT vao cDNA si i, cDNA si i c a vao trong cac plasmid vector qua phng thc ni dA:dT. Tuy

nhin, ui ng trung hp dA:dT it khi c dung tao dong cDNA, ly do chinh la khng co phng thc thich hp ct cDNA gn trong plasmid nh ui dA:dT.

1.2. ui dC:dG Phng php c s dung rng rai hn tao dong cac cDNA bng ui ng trung hp oi hoi b sung cac ui dC cho cDNA si i va cac ui dG c b sung vao plasmid vector a c ct han ch bng PstI. Enzyme PstI ct chui 5 CTGCAG3 tao ra u 3 tn cung la c cht ly tng cho vic b sung cac ui ng trung hp. Cac dong cDNA mang cac ui dC:dG co th d dang tach ra khoi plasmid bng cach thuy phn nh PstI (Hinh 6.5). S lng cac gc dA:dT va dC:dG cn thit cho hiu sut gn ti thich cDNA vao plasmid vector a c xac inh, s lng cac gc trn plasmid va cDNA phai xp x nhau, vi khoang 100 gc c b sung ti mi DNA ni dA:dT va khoang 20 gc ni dC:dG.

2. Cac linker va adapter nhn tao Cac linker cha mt hoc nhiu vi tri ct han ch cho php ni cDNA si i vi cac plasmid vector hoc bacteriophage vector. cDNA si i c x ly vi DNA polymerase cua bacteriophage T4 hoc DNA polymerase I cua E. coli, cac enzyme nay loai bo u tn cung 3 si n so le bng hoat tinh exonuclease 3 5 va lp y cac u tn cung 3-OH bi khuyt bng hoat tinh trung hp (polymerization). S phi hp cua cac hoat tinh nay a tao ra cac phn t DNA u bng, sau o cac cDNA nay c u vi mt s lng ln cac phn t linker vi s co mt cua bacteriophage T4 DNA ligase (enzyme xuc tac cho qua trinh gn cua cac phn t DNA u li vi linker) (Hnh 6.6).

Hinh 6.5. Tao dong cDNA si i bng ui ng trung hp dG:dC. Enzyme terminal transferase co hoat tinh tao nhom homopolymer u 3-OH cua DNA si i lam li ra mt u cui c cht cua no la DNA si n.

Hnh 6.6. Minh ha mt linker. (a) on 5-CCGGATCCGG-3 mang v tr nhn bit cho BamHI. (b) Linker ny c gn vo cDNA u bng nh DNA ligase. (c) Cu trc ny sau c ct bng BamHI to ra u 5 li.

Cac phn t DNA si i mang cac u kt dnh nhn tao c ct vi tri ct han ch trong linker, tinh sach, va sau o gn vi vector cung c ct bng RE tng ng tao ra cac u dinh tng ng vi cac u cua linker. Co th han ch s tai tao lai vong cua plasmid vector (khng tai t hp) bng cach x ly cac vector c ct bng enzyme phosphatase (calf intestinal alkaline phosphatase-CIAP) trc khi thc hin phan ng gn vi cDNA. Vic s dung adapter co hiu qua hn linker. Cac adapter co kich thc ngn, la cac oligonucleotide si i mang mt u bng ( gn vi cDNA si i) va mt u tn cung kt dinh ( gn vi u tn cung tng ng trong vector). Khng ging nh linker, cac adapter khng oi hoi phai ct bng cac RE sau khi chung c gn vi cDNA si i. Tuy nhin, cac phn t cDNA mang cac adapter c phosphoryl ha (phosphorylation) co th tao thanh cac phn t mach vong ong bng lin kt cng ha tri (dang khng th tao dong) hoc cac phn t mach thng dang kham (dang hon toan khng mong mun) trong sut phan ng gn tun t vi s co mt cua vector DNA a c dephosphoryl ha (Hnh 6.7).

Hnh 6.7. Minh ha mt adapter. (a) Adapter, c u tn cng 5 tng ng vi enzyme HindIII, c gn vo cDNA u bng nh DNA ligase. (b) u 5 ca adapter c th c dephosphoryl ha ngn cn s t kt ni li.

Khi ni cac linker hoc adapter vi cac phn t cDNA si i u bng, phan ng gn cn c tin hanh trong mt dung tich ti thiu ( duy tri mt nng cao cua linker/adapter). Nng phn t cua linker/adapter phai ln hn nng cua u tn cung cua cDNA it nht la 100 ln. Cui cung, trc khi oan cDNA c gn vao vector, cac adapter khng co phan ng va cac san phm co trong lng phn t thp (do phan ng ct han ch tao ra) cn c loai bo bng sc ky ct, in di agarose hoc polyacrylamide gel.

IV. Cac phng phap tao dong cDNA khac 1. Tao dong mRNA -cDNA Mt phng phap khac tao dong cDNA la bin nap vao E. coli cac th lai mRNA-cDNA a c gn vi cac plasmid vector. Cac vi khun vt chu loai bo mRNA va thay th no bng DNA. Sau khi si cDNA u tin c tng hp theo cach thng thng, cac gc dA c b sung cho th lai mRNA-cDNA sau o c u vi plasmid mang cac ui dT. Do u tn cung 3-OH cua RNA kem it nht 10 ln trong phan ng tng ng vi DNA nn hu ht cac gc dA c b sung cho th lai u phi hp u 3 cua DNA. Vic ni cac u khac cua th lai vi vector co kha nng thanh cng do mi lin kt hydrogen gia poly(A) t nhin u 3 cua mRNA va plasmid co ui dT. Phng php nay c mt s thun li sau: - Khng cn tng hp si cDNA th hai. - Ct vong cp toc DNA bng nuclease S1 la khng cn thit. Tuy nhin, han ch cua phng php nay la co hiu qua kem it nht 10 ln so vi phng phap tao dong cDNA si i va vi th khng thich hp cho vic xy dng mt s lng ln cac dong cDNA.

2. B sung tun t cac linker khac nhau cDNA si i, c tng hp bng c ch t mi (self-priming), c gn vao mt loai linker nhn tao trc khi vong cp toc trong cDNA bi ct. Vi th, cac linker chi c b sung mt u cua cDNA si i (u tng ng vi u tn cung 3 cua mRNA). Sau o cDNA c tinh sach khoi cac linker tha, vong cp toc c ct bng nuclease S1 va sa cha bng oan Klenow cua DNA polymerase I cua E. coli trc khi

loai linker th hai c gn vao cDNA. Cac linker nay se c gn vao ca hai u cua cDNA. cDNA c gn linker kep sau o c x ly vi cac RE thich hp va gn vao trong vector bng phng phap tao dong inh hng (Hinh 6.8). Phng phap nay c dung gn cDNA theo hng chinh xac cua cac promoter cho phep biu hin cac oan chn (inserted sequences) trong vi khun. Cac bacteriophage vector cho phep tao dong inh hng cung a c pht trin trong thi gian gn y.

3. Tao dong cDNA bng cac primer-adapter Ngay nay, vic san xut d dang cac oligonucleotide primer cua cac trinh t xac inh a cho phep phat trin cac phng phap tao dong s dung primer-adapter cha mt vung cua DNA ng trung hp u tn cung 3 va mt vi tri ct han ch u tn cung 5. Cac chui ng trung hp c dung lam mi tng hp si th nht va si th hai cua cDNA, va cac vi tri ct han ch bn canh c s dung a cac phn t cDNA si i cui cung vao trong mt vector thich hp. Trong m hinh n gian nht, phng phap nay cho phep cac cDNA c tao dong vi hiu sut cao (Hinh 6.9).

Hinh 6.8. To dong cDNA bng cach b sung tun t cac linker nhn tao. on Klenow to u bng cho cDNA si i v enzyme nuclease S1 ct vng cp tc. Cc linker th nht v th hai c gn tun t vo hai u ca cDNA, sau on cDNA ny s c ct cng enzyme hn ch vi vector to dng c v tr nhn bit trn hai linker nhn to. Cui cng, on cDNA c hai u tng ng c gn vi vector v bin np vo E. coli.

Hinh 6.9. Tng hp cDNA si i bng phng phap primer-adapter

oan cDNA c tng hp theo phng php trn khi gn vao trong cac vector biu hin nh gt20 va gt22 chi co 1/6 c hi biu hin trong vi khun, l do: chi mt na cac phn t cDNA c a vao trong vector theo hng chinh xac i vi promoter lacZ, va chi mt trong ba phn t c gn hng phai se trong khung oc chinh xac san xut protein hp nht. V. Cac bacteriophage vector dung trong tao dong cDNA 1. Cac bacteriophage vector c dung ph bin Hai loai vector biu hin ca bacteriophage thng dung xy dng th vin cDNA la gt10 va gt11. Vector gt10 dung xy dng cac th vin ch c sang loc bng cac mu do nucleic acid, trong khi vector gt11 dung xy dng cac th vin c sang loc bng cac mu do min dich phn lp cac chui DNA ma hoa cac khang nguyn c hiu.

1.1. Vector gt10

gt10 la vector c thit k nhn cac oan DNA ngoai lai trong vung ma hoa 2 cua gen c ch cI . Cac vector nay mang gen cI, ging nh bacteriophage , no tao thanh cac vt tan m uc trn hu ht cac chung E. coli. DNA cua gt10 mang vi tri nhn bit n EcoRI nm trong vung ma hoa cua gen cI la ni ma cac oan DNA co th gn vao. Kt qua chn oan DNA se lam bt hoat gen cI, san sinh ra cac bacteriophage cI tai t hp va tao thanh cac plaque mau sang, d dang phn bit vi cac plaque m uc cua gt10 b me. DNA cua gt10 b me xp x 43 kb va nh vy co th nhn cac oan DNA ngoai lai dai ti 7,6 kb. Cac th vin cDNA c xy dng trong gt10 lun cha hn hp cac bacteriophage tai t hp va khng tai t hp. Hai loai bacteriophage nay co th phn bit kiu hinh nh vao kha nng tao thanh cac plaque cua chung.

1.2. Vector gt11 gt11 la vector biu hin mang ban sao cua gen lacZ cua E. coli, co vi tri nhn bit n EcoRI nm vung ngc hng 53 bp cua codon kt thuc dich ma cua gen lacZ. DNA ngoai lai co kich thc xp x 7,2 kb co th c gn vi tri nay. Cac chui ma hoa gn trong khung oc theo hng chinh xac se c biu hin san xut cac protein dung hp (fusion protein) co u tn cung amino cha cac chui -galactosidase va u tn cung carboxyl cha mach polypeptide ngoai lai. Mt s protein dung hp nay se th hin cac yu t quyt nh khang nguyn (epitopes) c phat hin bi kha nng phan ng vi cac khang th cua chung. Cac protein dung hp mang cac yu t quyt nh khang nguyn ngoai lai co th c phat hin bng cach sang loc cac plaque vi mu d min dich. Bacteriophage o c dan trai 42 C trn E. coli. Sau khoang 4 gi, cac ia petri c chuyn n nhit o 37 C va c phu mang loc v trung nitrocellulose c thm isopropylthio--D galactoside (IPTG), cht lam bt hoat gen c ch lac va cam ng biu hin protein ngoai o lai. Sau mt vai gi nui 37 C, cac mang loc c u vi khang th lin kt vi khang nguyn quan tm. Cac khang th nay sau o c phat hin bng cac xt nghim hoa hoc phong xa (radiochemistry) hoc hoa hoc m (histochemistry).

2. Mt s bacteriophage vector khac 2.1. Vector gt18 v gt19 Hai loai vector gt18 va gt19 co ngun gc t vector gt11 c cai tin mang vng tao dong (polycloning sites) vi tri EcoRI n cua gt11. Co th s dung hai loai vector nay tao dong inh hng va khng inh hng cDNA. Hn na, it nht mt trong cac vi tri cua vung tao dong (SalI) it khi xut hin trong DNA ng vt co vu (trung binh 1 vi tri/oan 100 kb), va vi th it co kha nng cc dong cDNA co cha vi tri SalI. Nh vy, cac cDNA si i co u bng mang cac linker SalI khng cn phai c bao

v bng cach methyl hoa (methylation) trc khi c thuy phn bng RE. Khi cac oan linker c loai bo, se dn n kt qua qun th cac phn t cDNA co th c gn trc tip vi cac nhanh cua gt18 hoc gt19.

2.2. Vector gt20 v gt21 Cc vector gt20 va gt21 co ngun gc tng ng t gt18 va gt19, va nh vy co nhiu tinh cht ging nh a m ta gt18 va gt19. Nhng thay i c bit so vi gt18 va gt19 nh sau: - Vi tri tng hp chi c a vao vector cho phep loi b s chon loc oan chn cDNA cha cac vi tri chi. - Cac vi tri SacI va XbaI c loai bo khoi cac nhanh cua vector sao cho vi tri XbaI trong vung tao dong la duy nht, cac thao tac nay lm khuyt mt oan khoang 500 bp trong DNA ca bacteriophage phia bn phai gen lac5. c im nay cho phep nhn cac oan chn DNA ln hn (ti 8,2 kb) so vi cc vector gt18 va gt19.

2.3. Vector gt22 v gt23 Cac vector nay cung bt ngun t gt18 va gt19, mang trnh t tng hp chi va cc v tr tao dong trong mt khung khac gn u 3 cua vung ma hoa gen lacZ. Cc vector gt22 va gt23 ging ht nhau ngoai tr hng cua cc v tr tao dong. Cac vi tri XbaI va SacI cac nhanh cua gt18 va gt19 bi loai bo, nh vy cac vector ny chi mang nm vi tri ct han ch duy nht tao dong l: NotI, XbaI, SacI, SalI va EcoRI. Cac oan cDNA gn vao mt trong nm vi tri co th c biu hin nh la mt protein dung hp LacZ. Cc vector gt22 va gt23 c thit k cho phep tao dong inh hng cDNA vao trong cac vi tri SalI va NotI. Tng hp si th nht va th hai cua cDNA bng phng php primer-linker cho phep cDNA si i c ct bng SalI va NotI va gn trc tip trong cac nhanh cua vector theo hng lin quan vi promoter lacZ. Mt trong ba th tai t hp se mang phn t cDNA trong khung oc chinh xac san xut protein dung hp.

2.4. Vector ZAP Vector ZAP mang vung tao dong cung hng vi promoter lacZ cua E. coli. oan cDNA dai 10 kb co th c gn vao trong cc vi tri nay va biu hin hoc trong vi khun c xm nhim hoc trong cac th tim tan c cam ng, nh m ta gt11. Hn na, cac protein dung hp ZAP co th c biu hin t cac plasmid co s lng ban sao ln. Vector ZAP co mt s c im nh sau: - Co su vi tri RE trong vung tao dong c s dung tao dong inh hng cac phn t cDNA, trong o co mt vi tri ct EcoRI tng t nh gt11.

- Co cac promoter ca bacteriophage T3 va T7 nm bn canh vung tao dong. - Co cac trinh t co ngun gc t bacteriophage f1 chuyn i in vivo oan chn DNA t bacteriophage vector ti Bluescript plasmid, cac trinh t cua no c cha trong ZAP. Qua trinh ct bo c thc hin d dng nh s b tri cac trinh t cua bacteriophage f1 (khi u tng hp DNA si n) v mt phia cua Bluescript plasmid DNA mang vung tao dong va s b tri cac trinh t bacteriophage f1 (kt thuc tng hp DNA si n) v mt phia khac cua plasmid DNA. Vector ZAP la dang u tin cua vector th h mi c thit k nhm cung cp mt trinh t cac vi tri tim nng tao dong va biu hin cDNA, mt phng phap tin li v n gian thu hi va thc hin cc thao tac tip theo cua cDNA. Do tinh linh hoat cua chung, cac vector nay thay th cho gt10 va gt11 va c xem la vector thich hp xy dng cac th vin cDNA. Vector ZAPII cung tng ng vi ZAP.

VI. Nhn dang cac dong cDNA quan tm

1. Cac phng phap sang loc Co ba phng phap sang loc th vin cDNA cho cac dong quan tm: - Lai nucleic acid. - Phat hin cac khang nguyn min dich c hiu. - Chon loc sib cac dong cDNA. Di y chi trinh bay hai phng phap ph bin l lai nucleic acid v pht hin cc khng nguyn min dch c hiu.

1.1. Lai nucleic acid y la phng phap c s dung ph bin va ang tin cy nht khi sang loc cac th vin cDNA tim kim cac dong quan tm. Sang loc bng phng phap lai nucleic acid cho phep phn tich ng thi nhiu dong va nhanh, khng oi hoi cac dong cDNA phai hon chinh va san phm c tng hp trong t bao vt chu phai co hoat tinh sinh hoc hoc khang nguyn. Hn na, c s ly thuyt cua ky thut lai nucleic acid c hiu bit y u. iu nay cho phep phat trin mt s lng ln cac ky thut khac nhau co th iu chinh cac mu d ca nucleic acid co cac chiu dai va c im khac nhau. - Cac mu d tng ng. Cac mu d tng ng cha it nht mt phn cua chui nucleic acid chinh xac cua dong cDNA mong mun. Chung c dung trong nhiu trng hp khac nhau, vi du: khi mt dong b phn cua cDNA hin co c s dung phn lp mt dong hon chinh t th vin cDNA. Thng thng, oan bt ngun t mt u hoc u khac cua dong hin co c phn lp, anh du phong xa in vitro va dung

 thm do th vin. Lai vi cac mu d tng ng thng c tin hanh di cac iu kin nghim ngt (stringency). - Cac mu d tng ng tng phn. Cac mu d tng ng tng phn c dung phat hin cac dong cDNA co quan h ho hang, nhng khng ging ht nhau hon toan, vi cac trnh t ca mu d. Nu cac mu d khang th ln nucleic acid khng co sn, thi co th thay i bng mt vai phng php. Vi du: Nu dung gen a c tao dong t cac loai khac hoc nu gen lin quan a c tao dong t cung loai, thi cn tin hanh mt loat cac thi nghim kim tra xem co s bao toan y u cua trinh t nucleic acid hay khng cho phep sang loc th vin cDNA bng cach lai. iu nay c thc hin d dang nht bng cach t chc mt loat cac phan ng lai Southern va Northern cac mc nghim ngt khac nhau. Dung mt lng ti thiu (5-10 g) cua genomic DNA a c ct bng RE chay in di trn agarose gel 0,8%, cac phn oan sau o c chuyn vao mang lai nitrocellulose. Mang c ct thanh tng manh nho, mi manh c lai di cac iu kin khac nhau vi cac lng mu do co hoat tinh phong xa ging nhau. Mt lot cac phan ng lai tng t co th tin hanh vi cac mRNA c tiu phn hoa bng in di va chuyn ln gia th rn. Mc ch ca c hai trng hp la thit lp cac iu kin cho phep cac gen c tao dong trc o c s dung nh la mu d cho cDNA quan tm. - Cac oligonucleotide mu d nhn tao. Cac oligonucleotide mu d nhn tao la cac on nucleotide cua trinh t xac inh c tng hp in vitro. Trinh t cua cac mu d nay c suy lun, bng cach dung ma di truyn, t cac vung ngn cua chui amino acid a bit cua protein quan tm. Do s thoai bin (degeneracy) cua ma di truyn, chui amino acid xut c th khng c c trng chnh xc bi cc oligonucleotide n c d oan cho trinh t. Mc du, trong rt nhiu trng hp, trinh t cac amino acid ging nhau co th c c trng bi nhiu oligonucleotide khac nhau. Khng co cch nao bit chc chn cac oligonucleotide nay trn thc t c c dung trong gen quan tm hay khng. Ba giai phap c a ra cho vn nay l nh sau: + Mt ho oligonucleotide co th c tng hp cha tt ca cac trinh t co kha nng ma hoa cho mt trinh t a cho cua chui amino acid. S lng cac thanh vin trong ho nay tuy thuc vao mc thoai bin cua ma b ba cho cac amino acid c bit. Tuy nhin, do tt ca cac trinh t oligonucleotide co kha nng c hin din, nn it nht mt trong s cac thanh vin se tng hp vi dong cDNA quan tm. Duy tri kich thc cua mi ho trong ty l d s dung, cac oligonucleotide ngn (14-17 base) thng c s dung, mt kich thc ti thiu c tin hanh cho phan ng lai. + Oligonucleotide dai hn (40-60 base) cua trinh t duy nht co th c tng hp bng cach dung cac ma b ba c s dung ph bin nht cho mi amino acid (tranh dung dinucleotide CpG, vi no c hin din lai trong hu ht cac cDNA cua eukaryote). Tt nhin, oligonucleotide nay se khng tng hp mt cach chinh xac vi trinh t trong

cDNA, nhng no se c inh u tt phat hin bng cach lai di cac iu kin khng nghim ngt (non-stringency). + Mt oligonucleotide co th c tng hp cha mt base nh la inosine cac vi tri thoai bin tim tng. Inosine co th bt cp vi tt ca bn loai base truyn thng ma khng lam tn thng nghim trong kha nng n inh cua cac th lai co kt qua. Vi th, no co kha nng sn sinh ra cac ho oligonucleotide dai hn c lam giam s lng va cn lai vi gn nh tt ca cac dong cDNA co th ma hoa cho protein quan tm. Cui cung, nu trinh t protein co sn t u tn cung amino cua protein, thi th vin cDNA c sang loc phai co cht lng cao chc chn rng hu ht u tn cung 5 cua mRNA c ai din.

1.2. Phat hin min dich cac khang nguyn c hiu Cac th vin cDNA c xy dng trong cac vector biu hin nh gt11, gt18 23, ZAP... co th c sang loc bng khang th theo hng chng lai protein quan tm. Cac mang loc nitrocellulose in cac vt tan cua vi khun c ngm trong dung dich cha khang th. Sau khi ra, mang loc c u vi protein A cua Staphylococcus aureus hoc bng khang th th hai theo hng chng lai cac yu t quyt nh khng nguyn c hiu loai cua khang th th nht. cac m ta u tin cua phng phap nay, phi t 125 (ligand) th hai c anh du ng vi phong xa vi I. Ngay nay, phi t th hai c lin kt hoa tri vi enzyme ma hoat tinh cua enzyme co th c phat hin bng hoa t chc hoc (vi du: alkaline phosphatase). Chia kha thanh cng cua phng phap nay nm trong cht lng cua khang th. iu cn thit la khang th nhn din c protein bi bin tinh (Vi du: no phai cho cac tin hiu manh trong phn tich Western blot-xem chng 7). Hn na, qua trinh sang loc c tin hanh d dang hn va mn cam hn nu khang th c bt ngun t huyt thanh a dong (polyclonal antiserum) chun cao. Bi vi cac huyt thanh nh th phan ng binh thng vi nhiu yu t quyt nh khng nguyn khac nhau, c hi phat hin dong cDNA biu hin oan protein quan tm c tng ln. Cac huyt thanh a dong thng cha cac khang th phan ng cheo nhn din cac thanh phn khng tai t hp cua vi khun tim tan v chng phai c loai bo trc khi qu trnh sang loc c thc hin. Phan ng lin kt khng c hiu se thp hn nhiu khi s dung khang th n dong lam mu d. Tuy nhin, s lng cac th tai t hp co th c phat hin cung bi giam bi vi mi khang th n dong ring bit co th phan ng vi chi mt yu t quyt nh khng nguyn n. Vi th, mu d min dich ly tng co th cha mt hn hp cua nhiu khang th n dong khac nhau, ma mi khang th phan ng manh vi protein bi bin tinh.

2. Cac phng phap xac nhn cac dong cDNA Cac th vin cDNA thng c dan trai mt cao sang loc vi khang th hoc cac mu d ca nucleic acid, va mi dong phan ng dng tinh trong vong u tin oi hoi mt s chu ky dan trai va sang loc b sung trc khi chung c xem nh thun khit. Tuy nhin, kha nng phan ng thich hp vi mu d c bit la khng y u chng minh dong cDNA thu c bt ngun t mRNA quan tm. S chng minh chi tuyt i khi cho thy dong cDNA cha mt khung oc m ma hoa cho trinh t amino acid hon toan cua protein. Mt s vn sau cn c lu m bo mc chinh xac ca cc dng cDNA thu c: - Biu hin cua protein t cDNA hon chinh trong cac t bao prokaryote hoc eukaryote th hin cac hoat tinh sinh hoc hoc enzyme chinh xac. - S tng ng gia cac phn cua chui nucleotide cua cDNA va cac chui amino acid cua cac peptide co ngun gc t protein c tinh sach. - S tng ng gia cac ban peptide cua chui polypeptide c tng hp in vitro bng s phin ma cua dong cDNA va cac ban peptide cua protein ich thc. - S kt tua min dich cua polypeptide c tng hp in vitro hoc in vivo t s phin ma dong cDNA bng cac khang th c tng kh nng chng li protein quan tm. S cht che cua th nghim nay tng ln khi no c tin hanh bi mt chui cac khang th n dong xac nhn cac yu t quyt nh khng nguyn khac nhau trn phn t protein. - Kt tua min dich protein ich thc vi cac khang th tng kh nng chng li cac peptide tng hp ma cac trnh t cua chung c xac inh bi trinh t nucleic acid cua cDNA c tao dong.

VII. Xy dng th vin cDNA trong bacteriophage vector S dung th vin cDNA co hai u im sau: - Cac dong cDNA cha trinh t ma ha lin tuc cua mt gen. Nhiu gen eukaryote la gian oan, co cha nhiu oan intron. Sau khi ct tin thn mRNA (pre mRNA) va ni lai, cac oan intron a bi loai va mRNA hon thin (mature mRNA) co trinh t ma ha lin tuc c tao thanh. Do cDNA c phin m ngc t khun mu mRNA hon thin nn cac dong cDNA co th tng hp protein cn thit vi s lng ln nh mong mun. - Nhiu protein c tng hp vi s lng ln bi nhng t bao chuyn ha va nh vy trong cac t bao nay mRNA cua protein o se co ty l cao va th vin cDNA c tao ra t cac t bao nay se co nhiu cDNA ma ha cho cac protein tng ng. S

di dao cDNA ca mt vai loai mRNA nao o lam giam nhe ang k vic xac inh ung dong mong mun t th vin gen. Phng phap tng hp gen t mRNA ngay cang c phat trin, no kt hp vi cac phng phap khac cua sinh hoc phn t cho phep ng dung trong nhiu linh vc. Cc bc chnh ca qu trnh xy dng th vin cDNA nh sau:

1. Chon loc kich thc cua cDNA cDNA sau khi ct han ch c phn oan bng sc ky ct Sepharose loai b nhng linker tha va cac phn t cDNA co kich thc nho hn 500 bp. Ct sc ky thng co kich thc 27 0,3 cm thich hp cho vic phn oan cac cDNA.

2. Gn cac cDNA vi cac nhanh cua bacteriophage Thng thng cn phai tin hanh th mt loat cac phan ng gn va phan ng ong goi. Trong cac phan ng nay, mt lng khng i cua cac nhanh bacteriophage c gn vi cac lng khac nhau cua cDNA, muc ich la xac inh lng cDNA tao ra it nht la 6 5 10 bacteriophage tai t hp. Phan ng gn cn c thit k sao cho ti thiu mt bacteriophage tai t hp n se cha nhiu hn mt phn t cDNA bng cach dung ty l cua cac nhanh c phosphoryl ha va cDNA chi 5% bacteriophage c tai t hp. Nu s dung cac nhanh dephosphoryl ha, thi cac bacteriophage khng tai t hp bi c ch hiu qua, va vi th khng th xac inh lng cDNA cn thit tao ra mt qun th bacteriophage cha 5% th tai t hp. Vic s dung cac nhanh dephosphoryl ha hoc cac nhanh co u tn cung khng tng thich, tao ra bng cach lp y tng phn, c khuyn co s dng khi xy dng th vin cDNA trong cac bacteriophage nh gt11, gt18-23 va ZAP, la nhng vector khng co h thng cho phep chon loc da theo cac bacteriophage b me. S lng cac th khng tai t hp co th c giam xung khoang 100 ln bng cach dung cac nhanh dephosphoryl ha. Trong h thng nay, cac bacteriophage khng tai t hp tao thanh cac plaque mau xanh trn cac chung E. coli thich hp khi co mt cua IPTG va X gal, trong khi o cac plaque tai t hp la khng mau.

3. Phn tich cac oan chn cDNA Thu thp khoang mi hai plaque ca bacteriophage tai t hp va chun bi DNA ct bng RE thich hp. Phn tich kich thc cua cac on chn cDNA bng in di trn agarose gel 1%, dung DNA marker co cac oan t 500 bp ti 5 kb. Nu cac

bacteriophage tai t hp cha cac oan chn co kich thc khac nhau va nu kich thc trung binh cua chng xp x 1 kb hoc ln hn, thi co th tin hanh cac bc tip theo (vi du: tao ra th vin cDNA hon chinh). Nu kich thc trung binh cua cac oan chn nho hn 1 kb mt cach ro rt, thi cht lng cua th vin la khng cao. Trong trng hp nay cn quay lai phn tich cht lng cua mRNA khi u va cac phan ng tng hp cDNA.

4. Tao th vin cDNA hon chinh Nu s dung cac nhanh dephosphoryl ha xy dng th vin cDNA, thi tinh toan lng cDNA cho kt qua tng gp 10 ln s lng cua cac plaque khng tai t hp. Nu s dung cac nhanh phosphoryl ha, thi tinh toan lng cDNA cho kt qua qun th bacteriophage cha xp x 5% cac th tai t hp. Thit k mt phan ng gn quy m ln, tng s lng cua tt ca cac thanh phn mt ty l thich hp. Chc chn rng thit k phan ng gn i chng chi cha cac nhanh cua bacteriophage ma khng cha cDNA. ong goi tt ca cac hn hp gn bng cach thit k mt chui cac phan ng gn, mi phan ng cha 0,5 g cac nhanh cua bacteriophage . Gp cac tiu th ong goi va xac inh chun cua bacteriophage gc trn cac chung vi khun thich hp.

5. Khuch ai th vin cDNA - Vector gt10. thit lp mt s cung cp lu dai cua th vin, thi no cn phai c khuch ai bng s sinh trng trn chung E. coli thich hp (chng han chung BNN102) trn ia agar. Nu th vin chi c sang loc mt ln, bc khuch ai nay co th bo qua va cac bacteriophage co th c dan mong trc tip trn chung BNN102 mt mt ti u tin hanh sang loc. - Vector gt11 va cac dn xut cua no va ZAP, ZAPII. Cac th vin cDNA c xy dng trong cac vector biu hin gt11, gt18, gt20 va gt22 phai c khuch ai trn chung E. coli Y1090hsdR (hoc nu la vector ZAP thi trn chung BB4, vector ZAPII thi trn chung XL1-Blue). Cac chung nay la khng hon hao cho s ct han ch kim soat vt chu nhng lai mang mt h thng methyl ha hoat ng. Cac vi tri tim tang cho phn ng ct han ch bng h thng EcoK s c methyl ha trong sut qu trnh khuch ai sao cho, nu cn thit, cac th tai t hp co th c dung gy nhim chung E. coli kha bin-ct han ch. Trong sut qua trinh khuch ai, iu quan trong la c ch san xut cac protein c t tim tang bi cac bacteriophage tai t hp.

Ti liu tham kho/c thm

1. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA and Struhl K. 2002. Short Protocol in Molecular Biology. Vol 1 and 2. 5th ed. John Wiley & Sons, Inc. USA. 2. Brown TA. 2001. Gene Cloning-An Introduction. 4th ed. Blackwell Science, Oxford, UK. 3. Calladine CR and Drew HR. 1997. Understanding DNA: The Molecule and How It Works. 2nd ed. Academic Press, London, UK. 4. Glick BR and Pasternak JJ. 2003. Molecular Biotechnology: Principles and Applications of Recombinant DNA. 3rd ed. ASM Press, USA. 5. Maniatis T, Fritsch EF and Sambrook J. 1989. Molecular Cloning-A Laboratory Manual. Cold Spring Habor Laboratory Press, USA. 6. Ohman DE. 1989. Experiments in Gene Manipulation. Prentice Hall, Englewood Cliffs, New Jersey, USA. 7. Primrose SB, Twyman R and Old RW. 2001. Principles of Gene Manipulation. 6th ed. Blackwell Science, Oxford, UK. 8. Rapley R and Walker JM. 1998. Molecular Biomethods Handbook. Humana Press Inc. New Jersey, USA. 9. Surzycki S. 2000. Basic Techniques in Molecular Biology. Springer-Verlag, Berlin, Heidelberg, Germany. Vong cp toc: hinh dang cua phn t DNA hoc RNA c dung lam mi cho qua trinh tng hp si th hai. 2 cI repressor: gen c ch c sn phm protein lin kt vi vng operator hoc promoter ca gen v km hm phin m bng cch ngn chn s lin kt ca RNA polymerase.
1

Chng 7

Biu hin gen ti t hp trong Escherichia coli

Vector biu hin l vector c th mang cac gen ngoi lai mong mun cho php thc hin s phin ma cua cac ban sao c tao dong va s dich ma cac mRNA cua chung trong Escherichia coli. Nhng vector nh th c dung biu hin cac gen ca eukaryote trong E. coli hoc tng hiu sut cac san phm ca gen prokaryote. Mc d E. coli khng phi l mt vt ch l tng biu hin cc gen ca eukaryote do chng khng c kh nng sa i hu dch m (post-translation modification) cho cc chui polypeptide ca cc protein c cu trc phc tp. Tuy nhin, trong mt s trng

hp ngi ta vn c th s dng vi khun E. coli cho nhng protein c cu trc n gin hn. i vi nhng protein c cu trc phc tp, vt ch thng c s dng l cc c th eukaryote (v d: nm men Saccharomyces cerevisiae, nm mc Aspergillus niger) do chng c th hu dch m c. biu hin tt c cc gen ngoi lai trong E. coli phi bt u bng vic gn on gen ngoi lai vo trong vector biu hin (thng l plasmid). Vector ny phi c cc cu trc cn thit sau: - Trinh t khi u sao chep (ori) co th to ra nhiu bn sao trong t bo vt ch. - Cc trnh t m ha gen ch th chn lc (selectable marker) m bo duy tr vector trong t bo. - Mt promoter kim sot phin m (v d: lac, trp hoc tac) cho php sn xut mt lng ln mRNA t cc gen c to dng. - Cc trnh t kim sot dch m nh trnh t lin kt ribosome c b tr thch hp v codon khi u AUG. - Mt polylinker a gen ngoi lai vo trong mt hng chnh xc vi promoter. Ch khi c cu trc y nh th, cc vector biu hin mang gen ngoi lai mi c bin np vo chng E. coli thch hp. Chng ny m t cc phng php v cc plasmid vector c s dng sn xut cc protein dung hp (fusion protein) v cc protein nguyn th (native protein) trong vi khun. Cc protein dung hp c th c sn xut vi mt lng ln, d dng tinh sch v c th c dng gy ra mt p ng min dch. Cc protein nguyn th c th c sn xut trong vi khun bng cch gn mt promoter mnh v mt trnh t lin kt ribosome hiu qu ngc hng vi gen c to dng (vng 5 khng dch m). Thng thng, cc protein c biu hin mc cao trong cc th vi khng ha tan.

I. Sn xut cc protein dung hp 1. Protein dung hp Protein dung hp (protein lai) c m ha bi mt gen lai do s dung hp in vitro hai on gen khc nhau. V vy, protein dung hp s mang trnh t amino acid ca hai protein khc bit (Hnh 7.1).

Hnh 7.1. Protein dung hp. Bao gm gen c to dng (gen A) v mt gen khc ca vi khun (gen B), do protein dung hp c cha mt phn protein ca vi khun. SD: on Shine-Dalgarno.

S dung hp gen c thc hin bng cch gn phn m ha ca gen c to dng gn u cui 3 ca gen vi khun (v d: gen lacZ). Protein dung hp c nhng u im sau: - Protein dung hp thng c sn xut vi hm lng ln do s khi u phin m v dch m c iu khin bi cc trnh t tiu chun ca E. coli. - Dung hp cc trnh t ngoi lai vi cc gen ca E. coli thng cho kt qu cc sn phm n nh hn cc protein ngoi lai nguyn th. - Protein dung hp c khi lng phn t ln hn so vi hu ht cc protein ca E. coli v v th d dng nhn bit trong in di polyacrylamide gel ca protein. Bng protein dung hp c th c ct ra khi gel, ng kh (lyophilize) sau nghin thnh bt v dng nh mt khng nguyn. - Protein dung hp co th c tit ra mi trng nh biu hin cua vi khun nu gen eukaryote c gn vi trnh t ma hoa cho peptide tin hiu (signal peptide), khi peptide s nay tao ra s chuyn dich protein qua mang. Tuy nhin, hin tng nay chi xay ra mt vai trng hp. 2. Cc h thng vector biu hin cc gen dung hp vi gen lacZ Mt s h thng vector c pht trin biu hin cc gen dung hp vi gen lacZ. Chng hn, cc vector h pUR c cc v tr to dng BamHI, SalI, PstI, XbaI, HindIII v ClaI u 3 ca gen lacZ (Hnh 7.2). Bng cch chn la cc vector v v tr ct hn ch thch hp ngi ta c th tin hnh dung hp cho hu ht gen c to dng. Trong mt s trng hp, s dung hp c th thc hin bng cch loi b u tn cng li hoc lm y u tn cng b khuyt trc khi gn.

Mt h thng vector tng t khc cng c pht trin sn xut cc protein dung hp, l cc vector biu hin pEX1-3. Promoter PR c iu ha v cm ng trong cng mt kiu nh promoter PL ca bacteriophage . Cc vector pEX1-3 c cc v tr to dng mang cc im nhn bit EcoRI, SmaI, BamHI, SalI v PstI nm u 3 ca gen lacZ. Cc codon kt thc dch m v cc tn hiu kt thc phin m c t cng hng (downstream) vi v tr to dng (Hnh 7.3).

Hnh 7.2. Cc vector h pUR. y l cc vector dung hp vi gen lacZ, c cc v tr to dng BamHI, SalI, PstI, XbaI, HindIII v ClaI u 3 ca gen lacZ. Chn on DNA ngoi lai (cDNA) vo trong cc v tr to dng thch hp cho php sn xut protein dung hp ca galactosidase hot ng vi chui peptide c m ha bi DNA ngoi lai. Cc vector pUR278, pUR288 v pUR289 ch cha mt v tr PstI trong gen Ampr. Cc vector pUR290, pUR291 v pUR292 cha mt v tr PstI trong vng to dng do v tr PstI trong gen Ampr b

loi b. S dng cc plasmid vector ny rt tin li khi on DNA ngoi lai quan tm c to dng trong v tr PstI bng phng php ui GC.

Hnh 7.3. Vector pEX2. Plasmid vector ny di khong 5,8 kb c thit k biu hin on DNA ngoi lai (cDNA) c dung hp u 3 ca gen lacZ. Phn tn cng amino ca gen lacZ c thay th bng mt s trnh t ca gen cro ca bacteriophage v gen lacI ca E. coli. Promoter PR ca bacteriophage (dng biu hin protein dung hp) c iu ha bi gen c ch cIts857 ca bacteriophage . Vng to dng hin din u 3 ca gen lacZ, tip theo l cc codon kt thc dch m (Stop) v tn hiu kt thc phin m (Term) ca bacteriophage fd.

3. Xy dng plasmid biu hin v pht hin cc protein dung hp Gn plasmid vector (pUR hoc pEX) thch hp vi on DNA ngoi lai to ra mt s dung hp trong khung c. Bin np cc vector ny vo E. coli (chng K12 71/18 hoc JM 103 cho cc vector pUR, chng M5219 cho cc vector pEX). Kim tra cc khun lc n mang on DNA ngoi lai mong mun bng cch tch chit DNA

(DNA miniprep) ca plasmid vector, sau ct bng enzyme hn ch thch hp v in di kim tra trn agarose gel. Sng lc cc khun lc sn xut protein dung hp. Phng php sng lc khun lc pht hin protein dung hp c tin hnh nh sau: Nui cy qua m 37oC (mt lng nh) t 5-10 khun lc trong mi trng LB c cha ampicillin. Sau , cm ng nui cy bng cch b sung thm isopropylthio--galactoside (IPTG) cho vector pUR v tip tc nui 37oC, i vi vector pEX th chuyn nui cy 37oC ln nhit 40oC v tip tc nui cy (c sc kh). Sau cc khong thi gian nui cy khc nhau (1, 2, 3 v 4 gi), ly 1 mL dch nui cy ly tm nhanh thu tiu th, ti huyn ph chng trong m 1 SDS, bin tnh 100oC trong 3 pht, ly tm 12.000 g trong 1 pht nhit phng. Tin hnh in di SDS trn polyacrylamide gel 6%. Dng dch huyn ph t bo ch mang mt mnh vector lm i chng. Protein dung hp s xut hin nh l mt bng mi dch chuyn chm hn bng -galactosidase trong i chng. 4. Tch chit cc protein dung hp sn xut khng th
Protein dung hp c th c tch chit theo mt s cch: dng dch chit urea, sc k i lc aminophenylthiogalactoside, in di SDS-polyacrylamide gel hoc phi hp gia cc cch ny. Phng php n gin nht l in di SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis), nh trnh trnh by mc 3 (nui cy mt lng ln trong 200 mL). Nhum gel v pht hin bng protein mi, sau ct bng ny ra khi gel, ng kh khong 2 ngy ri nghin thnh bt mn. Bt ny s c dng tim vo th sn xut khng th.

II. Sn xut cc protein nguyn th Cc protein nguyn th c th c sn xut trong E. coli bng cch s dng promoter iu ha mnh v mt trnh t lin kt ribosome hiu qu. biu hin gen prokaryote c trnh t lin kt ribosome mnh ch cn cung cp mt promoter l . Trong khi , biu hin mt gen eukaryote (hoc mt gen prokaryote vi mt trnh t lin kt ribosome yu) cn phi cung cp c promoter ln trnh t lin kt ribosome (Hnh 7.4). Cc mc biu hin c th khc nhau t di 1% cho ti hn 30% protein ha tan tng s (total soluble protein) ca t bo.

Hnh 7.4. Protein nguyn th. Gen to dng (gen A) c t sau promoter v trnh t SD ca vi khun. mRNA ch m ha cc amino acid c hiu ca on chn to ra loi protein

nguyn th.

Biu hin ca cc gen prokaryote: Promoter Bc u tin khi biu hin cc protein ca eukaryote trong vi khun l chn mt vector biu hin mang promoter mnh (strong promoter) ca prokaryote. Promoter la trnh t DNA hng dn RNA polymerase lin kt vi DNA va khi u s tng hp RNA. Cac promoter khac nhau co hiu sut khac nhau, ni chung, promoter manh giup cac mRNA c khi u tn s cao. Cac promoter khac nhau u mang hai oan bao toan cao, mt c xac inh khoang 10 bp va mt khoang 35 bp nm vng ngc hng (upstream) vi im khi u cua s phin ma (cn gi l vng 5 khng dch m-5 untranslation region). Hai oan nay c nh gi rt quan trong trong vic xac inh cng ca promoter. Cac promoter thch hp nht cho biu hin cua gen ngoai lai E. coli la nhng promoter ma ca hai cng va kha nng iu ha th hin manh. Nu san phm cua gen c tao dong la c (toxin) i vi cac t bao E. coli thi sau o s nhn i gen se lam cho cng promoter yu va promoter khng iu ha c. S phin ma mc cao co th gy tr ngai cho vic sao chep DNA ca plasmid va dn n tinh khng n inh cua plasmid. Phn ny gii thiu cc vector biu hin mang promoter PL ca bacteriophage , promoter (lai) trp-lac v promoter bacteriophage T7. 1. Promoter PL cua bacteriophage Promoter PL cua phage (Hnh 7.5) la mt promoter manh, c kh nng iu ha tt v c dung trong mt s vector biu hin. nhit thp (31 oC), promoter PL c duy tri trang thai bi c ch bi san phm ca gen cI. Sau khi hoat tinh cua gen c ch bi pha hy bng cach tng nhit nui cy thi promoter PL s xc tin phin m phn ln mRNA. Mt vai vector s dung promoter PL ca nh: pPLa 2311, pPLa 8 v pKC30.

Hnh 7.5. Vector pKC30. pKC30 l plasmid c kch thc xp x 6,4 kb, mang promoter PL ca bacteriophage v v tr nhn bit HpaI nm vng cng hng (c 321 nucleotides) vi v tr khi u phin m ca PL. Plasmid ny l dng phn chia ca vector pBR322 v cha on HindIII-BamHI c ngun gc t bacteriophage c chn vo gia cc v tr HindIII v BamHI ca vector. on chn cDNA mang tn hiu promoter (PL), mt v tr c nhn bit bi sn phm ca gen N (nutL), bn thn gen N, v tn hiu kt thc phin m ph thuc rho (tL). V tr nhn bit HpaI nm vi vng m ha ca gen N. Cc trnh t DNA c chn vo trong v tr HpaI c th c iu chnh bng cch chuyn plasmid ti t hp vo th tim tan ca bacteriophage mn cm vi nhit ( cIts857). Cc t bo c sinh trng n gia pha log 30oC v sau thay i ti 40oC bt hot sn phm ca gen cI v m promoter PL. Vector ny c dng biu hin protein cII ca bacteriophage vi mt mc khong 4% protein ha tan tng s ca t bo.

2. Promoter trp-lac S biu hin cua cac gen c tao dong trong vi khun E. coli cn c iu ha bng cac promoter khac. Chng han promoter tac (mt dng promoter lai gia promoter trp v promoter lac) c s dng thnh cng sn xut mt lng ln protein trong E. coli (Hnh 7.6). - Promoter trp c iu ha bi gen c ch trp va co th c cam ng bi s b sung 3-IAA (3-indolylacetic acid) vao mi trng, hoc bng cach thiu tryptophan. - Promoter lac c iu ha bi gen c ch lac va vi th co th c cam ng bi s b sung nhn t cam ng IPTG cho nui cy vi khun.

- Cui cung promoter lai trp-lac cha oan trp-35 gn vi oan lac-10 va operator lac c Boer va cs thit k nm 1982, promoter lai trp-lac c iu ha bi gen c ch lac (lac repressor).

Hnh 7.6. Vector pKK177-3. pKK177-3 l mt tac vector cha cc v tr to dng gen ngoi lai cng hng vi promoter tac. Cng hng vi cc v tr to dng l rrnB mang gen 5S ca E. coli v hai nhn t kt thc phin m T1 v T2.

3. Promoter bacteriophage T7 Mt h thng biu hin khc c pht trin bi Tabor v Richardson (1985), Studier v Moffatt (1986) l h thng bacteriophage T7 RNA polymerase/promoter (Hnh 7.7). H thng ny c thit k cho cc biu hin c chn lc ca gen c to dng. Chng cho php mc biu hin cao ca mt vi gen khng c biu hin hiu qu trong cc h thng khc.

Hnh 7.7. Vector pET-3 mang promoter (P10) ca bacteriophage T7 10 v yu t kt thc phin m (T). Yu t kt thc phin m c th to ra cc th phin m c sc khng mnh hn i vi hot tnh exonuclease. Vector pET-3a l dng phn chia ca vector pET-3 trong vng khi u dch m (S10) ca bacteriophage T7 10 (protein chnh ca v bacteriophage T7) c mang v tr BamHI codon 11 c chn vo. V tr NdeI (CATATG) c t vng khi u dch m v c th c s dng xy dng plasmid biu hin cc protein nguyn th.

Hnh 7.8. Vector pAS1. Vector pAS1 l mt plasmid di khong 5,8 kb mang promoter P L ca bacteriohage v v tr ct hn ch duy nht BamHI nh v codon khi u ATG ca gen cII ca bacteriophage . Plasmid ny c ngun gc t pKC30, trong gen cII ca bacteriophage c chn vo v tr HpaI. Gen cII sau c ct b bi enzyme exonuclease cho ti khi ch cn li codon khi u ATG (G ca ATG l nucleotide u tin ca v tr BamHI). biu hin gen b mt codon khi u, pAS1 c ct bng BamHI v sau x l vi enzyme mung-bean nuclease hoc nuclease S1 loi b u li (u tn cng si n). Gn DNA u bng ny vi on DNA u bng bt u vi codon th hai ca gen c biu hin t gen trong khung vi ATG. Cc gen c chn vo theo kiu ny c iu ha bng cch chuyn plasmid ti t hp vo trong th tim tan mn cm nhit ca bacteriophage (cIts857). Cc t bo sinh trng ti pha log mun 30oC v sau nng ln 40oC bt hot gen c ch (repressor) v m promoter PL. Gen c chn vo cng c th c iu ha bng hot ng ca protein N nutL v nutR ngn cn kt thc tR.

Hiu sut dich ma cua mRNA chiu s chi phi cua mt vai yu t: - Mc b sung gia chui SD va u 3 cua 16S rRNA. - Khong cch gia chui SD va codon AUG chui DNA ca eukaryote nh v. - Nucleotide theo sau AUG anh hng s lin kt ribosome. a codon ATG vo trong gen c tao dong va duy tri vi tri lin kt ribosome (RBS) cua vi khun bng cach dung vector pAS1 (Hnh 7.8). Vector pAS1 mang promoter PL v RBS ca gen cII ca bacteriophage , codon khi u ATG c dung hp trc tip vi phn ma hoa u tn cng amino cua gen eukaryote mun biu hin.

Cch lm ny co th hn ch vic ti u khoang cach gia chui SD ca vi khun va codon khi u ATG cua gen eukaryote co c s biu hin hiu qua cua gen. oan DNA c gn promoter va chui SD sau o c bin np vo cc chng E. coli thch hp. Sng lc th bin np thu cc khun lc c mc phin m cao. III. Xc nh mc biu hin ca gen c to dng Ni chung c ba cch thng c dng nh gi mc biu hin protein ngoi lai ca gen c to dng: - in di polyacrylamide gel xc nh protein c kch thc thch hp c sn xut mc cao trong cc t bo mang vector biu hin. Thng thng, protein quan tm c th quan st bng cch nhum gel vi Coomassie Brilliant Blue hoc bng thuc nhum bc. Nu khng thy c bng protein mi khi dng cc thuc nhum ny, th nh du s trao i cht vi 100 Ci ca [35S]Met hoc [35S]Cys trn 1 mL dch nui cy trong 5 pht. in di SDS-polyacrylamide gel v thc hin phng x t ghi c th cho php pht hin protein quan tm.
- Phn tch Western blot bng cch dng cc khng th lin kt c hiu vi protein quan tm c thm tch ln mng nitrocellulose sau khi thc hin din di SDS-PAGE (xem chng 2).

- Nu mc biu hin thp th nn t gen lacZ cng hng vi gen c biu hin. Nh vy, nu s phin m hoc dch m hn ch biu hin ca gen th nhng thay i trong h thng biu hin c th c kim sot bng nhng thay i trong hot tnh ca -galactosidase.

1. Western blot Phn ng lin kt khng nguyn-khng th c tnh c hiu rt cao. V vy, c th p dng phn ng ny pht hin s c mt v tinh sch protein. Khng th (antibody) c sn xut khi a khng nguyn vo th v c tinh sch t mu th sau khi gy nhim. Nhng khng th to ra bng cch ny l nhng khng th a dng (polyclonal antibodies-do cc t bo lympho khc nhau tit ra), do chng c kh nng nhn bit mt s khng nguyn. Ngc li, khng th n dng (monoclonal antibodies) ch tng tc vi mt khng nguyn nht nh.

Hnh 7.9. S k thut Western blot

Khng th c nh du bng bng enzyme (hoc bng cht pht hunh quang) pht hin protein c hiu (thng c thm tch ln mng nitrocellulose sau khi chy in di SDS-PAGE, v c nh ) thng qua k thut Western blot (hoc immunoblot) (Hnh 7.9). C ch ca phn ng lin kt khng nguyn-khng th c trnh by hnh 7.10. Sau khi protein trn mng lai gn vi khng th th nht c hiu v tip n l khng th th hai c nh du enzyme (alkaline phosphatase, horse-radish peroxidase) th phc hp ny s c lin kt vi c cht to mu. S hin din ca protein ngoi lai (sn phm dch m ca gen ngoi lai c chuyn vo t bo vt ch) s c pht hin nh s xut hin mu ca phn ng lai.

Hnh 7.10. S m t lin kt protein (khng nguyn) vi khng th th nht c hiu v khng th th hai c nh du enzyme trong Western blot

S phn b ca protein c hiu trong t bo v t chc m cng c th pht hin bng k thut lai in situ (in situ hybridization) vi nguyn tc tng t Western blot. Ngoi ra, khng th c s dng tinh sch protein c hiu bng kt ta min dch hoc sc k i lc (affinity chromatography). Khng th nh du c dng nh lng khng nguyn trong k thut xt nghim hp th min dch lin kt enzyme (enzyme-linked immunosorbent assay) gi tt l ELISA.

2. ELISA ELISA l mt k thut ha sinh, cng da trn phn ng lin kt khng nguynkhng th, c dng ch yu trong min dch hc pht hin s c mt ca khng th hoc khng nguyn trong mu. Tng t k thut Western blot, trong ELISA ngi thng dng hai loi khng th. Mt khng th c hiu vi khng nguyn protein (khng th th nht). Mt khng th khc, phn ng vi cc phc hp khng th-khng nguyn, c kt hp vi enzyme (khng th th hai). Khng th th hai nh c lin kt vi enzyme (nh tn ca k thut xt nghim) nn c th bt mu vi c cht to ra tn hiu (Hnh 7.11).

Hnh 7.11. S k thut ELISA

Do ELISA c th thc hin nh gi s c mt ca khng nguyn hoc khng th trong mu bng phng php quang ph (o hp th quang), cho nn n l cng c hu ch xc nh nng (nh lng) khng nguyn v khng th.

IV. Tng mc biu hin ca gen c to dng Cac mc biu hin cua gen ngoai lai trong E. coli thng c kim tra bng th nghim chc nng hoc bng cac xt nghim min dich khac nhau. Tuy nhin, nu c at n mc ti a s biu hin cua san phm gen thi cac phn tch nh th la phc tap. khc phuc kho khn nay khi san phm gen eukaryote khng dung hp c biu hin ngi ta a s dung phng phap cho DNA ngoai lai c dung hp vi oan tng

ng cua gen lacZ cho php gen ngoi lai sao chep lai va dich ma mt cach hiu qua. Plasmid c dng trong phng php ny l pLG mang gen lacZ ma hoa u tn cng carboxyl c hot tnh -galactosidase. Tuy nhin, -galactosidase vn cha c tng hp do cn thiu promoter v trnh t SD plasmid. Do , mt promoter di ng (portable promoter) s c gn pha trc gen dung hp va iu chnh s biu hin mc cao protein dung hp co hoat tinh -galactosidase. Cac plasmid c cac oan promoter c t ti u co th nhn bit bng cach bin nap vi khun Lac va thu c hoat tinh -galactosidase trn cac ia chi thi lactose (lactose indicator). Cac plasmid nay sau o co th c dung tai cu truc gen eukaryote khng hp nht ma no c biu hin mc cao. Mc biu hin thp ca gen c to dng c th l do RNA khng n nh, s kt thc cha hon chnh, s dch m khng hiu qu, hoc chnh protein khng n nh. V. Tinh sch protein 1. Th vi v phng php ha tan th vi 1.1. Th vi Protein c mc biu hin cao trong E. coli thng to ra cc ht nh khng ha tan trong t bo cht (th vi), c th quan st bng knh hin ngc pha v tch khi dch tan ca t bo sng bng phng php ly tm. Cc t bo biu hin mc cao cc protein ngoi lai c c li bng ly tm v ph v bng cc k thut c hc, siu m, hoc dng lysozyme kt hp vi cht ty. Cc th vi (inclusion) c to tiu th bng ly tm v ra vi Triston X-100 v EDTA hoc urea. thu c cht ha tan, protein hot ng, cc th vi c ra phi ha tan v sau phi c ti cun xon. Mi protein c th cn mt phng php ha tan khc nhau v thng c xc nh theo kinh nghim. Cc iu kin khc nhau (v d: guanidine HCl [5-8 M], urea [6-8 M], SDS, alkaline pH, hoc acetonitrile/propanol) c th c s dng ha tan cc th vi. Trong nhiu trng hp, ch cn pha long n gin dch chit ha tan trong mt m thch hp l . Nu protein cha cc cu ni disulfide, ngi ta cn phi tin hnh oxy ha v kh glutathione. Mt i khi cn b sung ng dung mi (co-solvent) nh PEG, hoc cc cht ty ra nh Triton X-100, Tween 20 hoc Zwittergent 3-16. Sau khi ha tan thnh cng, c th th nghim cc phng php ti cun xon khc nhau bao gm s pha long hoc thm tch. Hiu sut to ra protein hot ng hoc protein c cc lin kt disulfite ging nh protein gc ph thuc vo nng , tinh sch v kch thc ca chui polypeptide; pH v cng lc ion ca dung mi;

v t l ti cun xon ca chng. Cc nhn t khc bao gm s lng ca cc lin kt disulfite v bn cht ca chnh protein. Trong mt s trng hp, cc protein ha tan c th rt kh ti cun xon v ngi ta thy rng vic b sung chaperonins c th gip ch cho cc qu trnh ny. Chaperonins l cc protein cn m bo cun xon chnh xc cc in vivo protein, vi kh nng thun li ca chaperonin ti t hp chng c dng xc tc cho qu trnh ti cun xon chnh xc ca cc in vitro protein. Nguyn nhn to thnh cc th vi cha c bit y , v khng phi tt c protein biu hin mc cao u to thnh th vi. T bo vt ch cng th hin mt vai tr quan trng. Cu trc chnh xc ca protein ti t hp cng c th nh hng s to thnh cc th vi. Mt nghin cu c thc hin vi -interferon ngi ti t hp cho thy ch mt vi thay i amino acid cng c th nh hng n s chuyn ha gia cc biu hin ca protein ha tan v khng ha tan trong E. coli. 1.2. Phng 1.2.1. Lm tan t bo php ha tan th vi

Ly tm thu tiu th t bo E. coli, ti huyn ph tiu th bng phenylmethylsulfonylfluoride (PMSF) v lysozyme, t nhit phng (RT) khong 20 pht (thnh thong khuy). Sau , b sung deoxycholic acid, t 37oC v khuy cho ti khi dch tan tr nn sn st, b sung DNase I v 30 pht RT.

1.2.2. Tinh sch v ra th vi - Phng php 1. Ly tm dch tan thu c bc trn, ti huyn ph tiu th bng Triston X-100 v EDTA, gi RT 5 pht. Ly tm 15 pht ly tiu th v ti huyn ph trong nc. Trn dung dch vi m 2 SDS gel-loading dye v phn tch bng in di polyacrylamide gel xc nh protein quan tm c trong tiu th khng. - Phng php 2. Ly tm dch tan, ti huyn ph tiu th bng nc. Sau , ly tm li v ti huyn ph tiu th trong Tris.HCl c b sung urea. Ly tm v ti huyn ph tiu th bng nc. Trn dung dch vi m 2 SDS gel-loading dye v phn tch in di polyacrylamide gel xc nh iu kin ra thch hp cho protein. 1.2.3. Ha tan cc th vi Treo cc tiu th c ra trong m dung ly cha PMSF v urea, 1 gi RT. B sung dung dch c KH2PO4, EDTA v NaCl 30 pht RT, duy tr pH 10,7 bng KOH. iu chnh pH ti 8,0 bng HCl 30 min RT. Ly tm thu tiu th v ti huyn ph bng m 1 SDS gel-loading dye. Phn tch in di xc nh mc ha tan.

2. Cc ui i lc Khi nim ui i lc xut hin khi thit k di truyn vi mc ch gip cho s tinh sch protein hoc enzyme hiu qu hn. Gen ca protein quan tm c dung hp vi chui DNA m ha cho mt s trnh t amino acid s n gin ha qu trnh tinh sch protein, bng cch bin i cc tnh cht ca n trong mt kiu c th d on. Mt trong cc trng hp u tin l dung hp di truyn ca mt s gc arginine vi C-terminus ca urogastrone. Gen ny s sn xut mt protein lin kt mnh vi khun trao i ion cho cation. ui polyarginine sau c loi b bng cch dng enzyme bt ng carboxypeptidase A.

Hnh 7.12. Tinh sch protein ch bng sc k i lc. Cho hn hp protein ti t hp i qua ct sc k c cha mt phi t lin kt c hiu vi cc protein mang mt ui i lc (v d: His, Histidine hoc GST, glutathione-S-transferase). Cc cht bn s c ra tri khi ct, v cc protein c lin kt trn ct sau s c ra gii dng tinh sch. ui i lc c nhiu u im trong tinh sch protein. Trong IMAC (immobilized metal ion affinity chromatography), His s lin kt chn lc cao vi Ni2+ hoc cc kim loi chuyn tip khc c bt ng trn phi t, protein c gn ui i lc c th c ra gii chn lc bng imidazole. Protein c gn ui GST lin kt vi glutathione nh mt phi t, v c ra gii vi cc dung dch glutathione. Cc protein c ui dung hp GST mang hot tnh enzyme

ch c th c tinh sch di cc iu kin t nhin. Ngc li, cc protein gn ui His c th c tinh sch di cc iu kin t nhin hoc bin tnh.

Hnh 7.13. Phn tch SDS-PAGE v nhum bng Coomassie Blue cc sn phm protein sau khi tinh sch bng sc k i lc. 1: Chun khi lng phn t ca protein. 2: Protein ha tan tng s. 3: Protein dung hp (protein ch v GST) c ra gii khi ct glutathione sepharose. 4: Protein dung hp c ct bng PresCission Protease cho ra 2 bng l GST v protein ch. 5: Protein ch c tinh sch sau khi cho i qua IMAC.

Kh khn ch yu cc dung hp i lc tinh sch protein l phi loi b thnh cng ui i lc v cc tc nhn c s dng. Vn ny c th c gii quyt tng phn bng s bin np di truyn cc im c hiu cao cho protease hoc cho s ct b cc lin kt acid khng bn ch ni ca protein ti t hp v ui i lc. Nhiu phng php phn ct c gi . S dng cc protease c hiu l thch hp hn c, bi v c th ng dng trong cc iu kin nh nhng, ch yu l thrombin, enterokinase v Factor Xa. Tt c nhng protein ny hot ng 37oC v c th phn ct cc v tr bn trong protein quan tm, hoc do mt tnh c hiu hoc t s nhim bn cc protease. Cui cng, cc bc sc k tip theo s c yu cu loi b ui i lc c phn ct v protease. Trong s pht trin gn y ca lnh vc ny, ngi ta s dng dng ti t hp ca protease 3C t rhinovirus ca ngi. Protease ny c kch thc nh (20 kDa) v c mt tnh c hiu rt hn ch. N c biu hin nh l mt protein ti t hp c dung hp vi glutathione-S-transferase. iu ny c mt vi u im, bn thn protease c th c tinh sch bng sc k i lc trn glutathione-Sepharose. Nu protein ch c biu hin nh l mt s dung hp vi glutathione-S-transferase th

n c th c tinh sch trn ct glutathione-Sepharose. Sau khi x l vi protease, protein ch c th c phn tch khi ui glutathione-S-transferase v protease bng cch chuyn qua ct glutathione-Sepharose th hai. Mt cch khc, phn ng phn ct c th c t trn ct glutathioneSepharose th nht bng cch b sung protease vo m ca ct, v th trnh c s cn thit cho mt ct th hai. Protease ny c sn dng thng mi di tn PreCission Protease do Amersham Pharmacia Biotech sn xut (Hnh 7.12 v 7.13).

Tai liu tham kho/c thm

1. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA and Struhl K. 2002. Short Protocol in Molecular Biology. Vol 1 and 2. 5th ed. John Wiley & Sons, Inc. USA. 2. Maniatis T, Fritsch EF and Sambrook J. 1989. Molecular Cloning-A Laboratory Manual. Cold Spring Habor Laboratory Press, USA. 3. Ohman DE. 1989. Experiments in Gene Manipulation. Prentice Hall, Englewood Cliffs, New Jersey, USA. 4. Primrose SB, Twyman R and Old RW. 2001. Principles of Gene Manipulation. 6th ed. Blackwell Science, Oxford, UK. 5. Rapley R and Walker JM. 1998. Molecular Biomethods Handbook. Humana Press Inc. New Jersey, USA. 6. Rosenberg IM. 1996. Protein Analysis and Purification-Benchtop Techniques. Birkhuser, Boston, USA. 7. Surzycki S. 2000. Basic Techniques in Molecular Biology. Springer-Verlag, Berlin, Heidelberg, Germany. 8. Walker JM. 2002. The Protein Protocols Handbook. 2nd ed. Humana Press Inc. Totowa, New Jersey, USA.