Phng php PCR

GVHD: TS. Nguyn Minh Tr PHN I: GII THIU V PHNG PHP PCR

Vic trin khai cc ng dng ca Sinh hc phn t thng vp phi tr ngi v s lng vt cht di truyn cn c. Cc phng php to dng in vivo tuy gii quyt c vn v s lng nhng i hi thao tc phc tp v thi gian di. S ra i ca nhiu phng php khuch i in vitro c chn lc, trong ni ting nht phi k n phng php PCR, o ln nguyn tc ca vic to dng c in, m ra nhng trin vng ng dng to ln. PCR c pht minh bi Kary Mullis (M) nm 1985, ban u k thut ny cn phc tp v t tin nhng nh nhng pht hin mi v Enzyme Polymerase chu nhit v my lun nhit cng nh ci tin quy trnh m phng php ny tr nn n gin hn, chnh xc v r tin hn, chnh v vy m n c s dng hu ht cc phng sinh hc phn t trn th gii. PCR l phn ng nhn to tng hp v khuch i mt on DNA nm gia hai trnh t gi l mi (Primer) to thnh hng triu bn sao ca on DNA nhm phc v cho nhng nghin cu pht hin, nh lng, chun on, di truyn PCR v cc p dng ca n hin nay trong nhiu lnh vc v ang lm c nhng kt qu tht s k diu, lm cho cc cng trnh nghin cu sinh hc phn t tr nn nh nhng v d dng hn gp nhiu ln so vi trc kia. Nu trc y mt th nghim sinh hc phn t phi ko di hng tun, thm ch hng thng, th nay cng th nghim c cng mc ch nh vy, vi PCR ch thc hin trong vi ngy. Nu trc y c nhng mc ch th nghim khng th thc hin c, th ngy nay vi PCR mc ch ny li c th thc hin c mt cc d dng. Chnh nh PCR m ngy nay, sinh hc phn t lm c nhng bc tin nhy vt trong mi lnh vc. C th ni rng PCR thc s lm mt cuc i cch mng trong sinh hc phn t.

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Phng php PCR

GVHD: TS. Nguyn Minh Tr PHN II: NI DUNG PHNG PHP PCR

1. Khi nim PCR (Polymerase Chain Reaction) l phng php invitro tng hp v khuch i mt on DNA c th nh cng hiu ca 2 mi oligonucleotide gn vo 2 si i ca on DNA ch vi s tham gia ca DNA polymerase. 2. Lch s pht trin. 2.1 Lch s ra i phng php PCR Phng php cn bn chy PCR c Kary Mullis v cng s pht minh nm 1985, ng ot gii Nobel v Ha hc vo thng 10 nm 1993 cho thnh tu ny. kin ca Mullis l pht trin mt quy trnh m DNA c th nhn ln nhiu ln mt cch nhn to qua nhiu chu k sao chp bi enzyme DNA polymerase. DNA polymerase c t nhin trong sinh vt sng, ni m n thc hin chc nng nhn DNA khi t bo phn chia. N lm vic bng cch ni vi si DNA v to si b sung. Theo quy trnh PCR gc ca Mullis, enzyme phn ng nhn bn DNA c thc hin
Kary Mullis

trong ng nghim (in vitro). Si DNA i b tch thnh 2 si n khi un nng 96C. Tuy nhin, nhit ny DNA polymerase b ph hy v vy cn b sung enzyme sau mi giai on nung nng ca mi chu k. Quy trnh PCR gc ca Mullis khng c hiu qu cao v n mt nhiu thi gian, cn mt lng ln DNA polymerase, v phi lin tc lu sut trong qu trnh PCR. Sau , quy trnh gc ny c pht trin bng cch dng DNA-Polymerase ly t vi khun thermophilic (a nhit) sng trong mch nc phun nhit trn 110C. DNA polymerase t sinh vt ny l thermostable (n nh nhit cao) v khi dng trong PCR n khng b ph v khi hn hp c nung nng tch si DNA. T , khng cn phi thm DNA-polymerase vo mi chu k, qu trnh sao chp DNA c th n gin v t ng hn. Mt trong nhng DNA-polymerase chu nhit u tin c phn lp c t Thermus aquaticus v c gi l Taq. Taq polymerase c dng rng ri trong thc nghim PCR (5/2004). Nhc im ca Taq l thnh thong n nhm ln trong qu trnh sao chp DNA, dn n t bin (sai) trong chui DNA, v n thiu tnh sa sai exonuclease 3-5. Cc polymerase nh Pwo hay Pfu, c phn lp t Archaea c c ch sa sai (c ch kim tra li sai) v c th lm gim mt cch ng k s t bin xy ra trong chui DNA c sao chp. Ngy nay,

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Phng php PCR

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s kt hp gia Taq v Pfu c th cung cp c tin cy cao ln s nhn bn chnh xc ca DNA. PCR c dng khuch i mt on DNA ngn, xc nh c mt phn. c th l mt gen n, hay mt phn ca gen. Tri vi sinh vt sng, quy trnh PCR c th copy mt mnh DNA ngn, c th ln n 10kb (kb = kilo cp base=1000 cp base). DNA l mt si i, v v vy n c o vi DNA b sung gi l cp base. Mt vi phng php c th copy mt mnh kch thuc ln n 40kb t hn nhiu so vi nhim sc th DNA trong t bo eukaryote v d nh t bo ngi cha hn 3 t cp base. Nh thc hnh hin nay, PCR cn rt nhiu thnh phn. Nhng thnh phn l: + DNA mu (template) cha mnh DNA cn khuch i + Cp mi (primer), xc nh im bt u v kt thc vng cn khuch i + DNA-Polymerase enzym xc tc cho vic nhn ln ca DNA. + Nucleotides (v d dNTP)l nguyn liu cho DNA-polymerase xy dng DNA mi. + Dung dch m, cung cp mi trng ha hc cho DNA-polymerase Phn ng PCR c thc hin trong chu k nhit. y l my un nng v lm ngui trong ng phn ng nhit chnh xc cho mi phn ng. ngn nga s bay hi ca hn hp phn ng, phn np y ca my PCR cng c un nng, trng hp lng dung dch phn ng qu t, ngi ta cho mt lp du (natural oil) ln trn b mt hn hp phn ng. 2.2 Hai bc tin quan trng a PCR n cuc i cch mng sinh hc phn t 2.2.1 S pht hin ra cc men polymerase chu nhit Chnh vic pht hin c cc men polymerase chu nhit l bc tin u tin lm th nghim PCR tr nn n gin hn. Chng ta th tng tng nu khng c men polymerase chu nhit th th nghim PCR s phc tp v km hiu qu nh th no khi m c sau mi chu k nhit li phi thm men polymerase mi vo v men c b hy bi nhit trong chu k nhit trc ? Cng nh s s dng cc men polymerase chu nhit m giai on bt cp ca on mi vo nucleic acid ch c c hiu hn v c thc hin nhit cao hn, do phn ng PCR tr nn c hiu hn. Cho n nay, c hng chc loi polymerase chu nhit c pht hin v tng hp ra. C nhng men polymerase trch t cc vi khun chu nhit nh Thermus aquaticus (Taq polymerase), Thermus thermophilus (rTth), Thermus lithoralis (Vent), Pyrococcus furiosus (Pfu),. C nhng men polymerase chu nhit c tng hp t cc vi khun khng chu nhit nhng mang gen ti t hp t cc vi khun chu nhit nh Ampli Taq, Vent (exo),. C nhng men polymerase chu nhit c hot tnh sa sai (proofreading) khi tng hp chui nucleic acid b sung (3-5exonuclease) nh

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Phng php PCR

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Ultima, Vent, Deep Vent, Pfu,. Nh vy m ngi dng c rt nhiu chn la s dng cho ng mc ch ca mnh.

Thermus aquaticus 2.2.2 My chu k nhit Trc y, khi cha c my chu k nhit, th nghim PCR c thc hin bng cch lin tc chuyn cc ng nghim phn ng PCR vo cc my cch thy c nhit khc nhau to ra c cc chu k nhit cho phn ng. Sau cng vic bng tay nhm chn v nng nhc ny c thay th bng cc robot tng i cng knh v t tin. Ngy nay, th nghim PCR c thc hin trong cc bung PCR ca my chu k nhit, cn gi l my lun nhit. Mt cch tng qut, my lun nhit gm cc b phn chnh nh sau : my thc hin.

Thermus thermophilus

My PCR

+ Mt bn phm n gin lp cc chng trnh chu k nhit v ra cc mnh lnh + Mt b vi x l ghi nh cc chng trnh np vo my, thc hin cc mnh lnh n bung PCR, cng nh ghi nhn cch thay i nhit bung PCR trong cc chu k nhit iu chnh cho ng vi chng trnh ang c thc hin. + Bung PCR l ni m cc chu k nhit c thc hin qua s iu khin ca b vi x l. Trong cc chu k nhit, nhit trong bung PCR c th a ln cao hay h xung thp trong mt thi gian rt ngn. lm thay i c nhit trong bung PCR, c hai phng php:

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Phng php PCR

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(1) Lin tc thi ln bung nhng lung kh nng sinh ra t mt n pht nhit, hay lung kh lnh sinh ra t mt dn lnh ca my lm lch. Vi phng php ny, my lun nhit hy cn tng i cng knh, kh t tin v khi my hot ng m thanh cng kh n o. (2) Phng php th hai hot ng da theo hiu qu Peltier ngc. Hiu qu Peltier l nguyn tc ca cc my o nhit in t : Khi p hai mnh kim loi vo nhau v khi c s cch bit nhit gia hai mnh kim loi th s sinh ra mt dng in v chnh s lu thng ca dng in ny khi o lng ra s phn nh s cch bit nhit gia hai mnh kim loi. Hiu qu Peltier ngc li vn hnh theo kiu ngc li, ngha l khi to ra mt dng in gia hai mnh kim loi th s to ra c mt s cch bit nhit gia hai mnh : bn ny nng, bn kia lnh. Khi thay i chiu dng in th nhit ca hai mnh cng thay i ngc li : bn ny lnh, bn kia nng. Cc my chu k nhit hin nay u c ch to da theo phng php th hai ny, nh vy m my tr nn rt gn nh, gi thnh r hn gp nhiu ln so vi trc y. 3. Nguyn tc ca phn ng PCR K thut tng hp DNA ngoi c th cng tun th nhng nguyn tc c bn ca sao chp DNA trong c th nh: on DNA cn nhn bn phi m xon thnh 2 mch n, cn c cc cp mi (mi xui, mi ngc), cn nguyn liu, iu kin mi trng thch hp v enzym DNA polymerase. Tuy nhin k thut PCR c khc l dng nhit cao (94oC) tho xon thay cho helicase kt hp vi enzym DNA polymerase chu nhit v h thng iu nhit cho tng giai on phn ng tng hp cng vi cc on mi c thit k ch ng. Nh k thut PCR m vi mt lng nh DNA ban u chng ta c th thu c lng DNA cn thit tin hnh cc th nghim v DNA. Mt phn ng PCR l mt chui phn ng gm nhiu chu k ni tip nhau, mi chu k gm ba giai on: Giai on bin tnh, giai on bt cp v giai on ko di chui. Giai on bin tnh (denaturation): Tch chui ADN t mch i thnh dng mch n. Phn t DNA si i c hnh thnh bi mi lin kt hydro gia hai mch n. Trong qu trnh bin tnh, nhit phn ng gia tng ln nhanh chng n 95oC v gi nhit ny trong khong 15-50 giy. nhit ny mi lin kt hydro gia hai mch n ca phn t DNA si i b ph v. Trong giai on bt cp tip theo, nhit phn ng gim nhanh chng lm cho mi lin kt hydro ban u khng hnh thnh li c kt qu lm to thnh hai phn t DNA mch n.

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Phng php PCR

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Mt s ch trong giai on bin tnh: 1. Vic tng nhit hoc ko di thi gian bin tnh c th lm tng tnh c hiu ca PCR nhng lm gim tui th ca enzyme DNA polymerase. 2. i vi nhng enzyme DNA polymerase bn nhit thng nn s dng nhit bin tnh 95C-98C. Bao gm bc bin tnh di lc khi u v mt bc bin tnh ngn trc tng chu k nhit. 3. i vi cc genome ln nh thc vt thng s dng bc bin tnh di n 5pht. lm gim nh hng i vi enzyme th c th b sung enzyme DNA polymerase sau khi bin tnh ban u. 4. Khi ti u PCR th cn gim ti thiu thi gian bin tnh mi chu k tng lng sn phm. Thng thng t 10s n 30s. 5. Tc gia nhit v ch iu khin nhit mi h thng lun nhit (PCR machine, thermocylcer) l khc nhau ph thuc hng sn xut. Mt s h thng cho php thay i thng s ny khi ti u PCR. Giai on bt cp (anealation): Gn cp mi c trng theo nguyn tc b sung. Trong giai on bt cp, nhit lai thng duy tr khong 50- 60 0C, trong thi gian 1560 giy. Qu trnh ny gip cho cc mi (primer) tm kim v bt cp b sung c hiu vi trnh t ch (DNA hoc cDNA) ti v tr mong mun. Nhit bt cp ph thuc vo s lng G,C c trong mi (primer).

Nhng ch trong giai on bt cp:

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Phng php PCR

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1. i vi primer di hn 22 nucleotide, nhit bt mi thng hot ng Tm thp nht + 3C. 2. i vi primer nh hn 22 nucleotide, nhit bt mi thng thp hn Tm thp nht, c th dng grandient nhit (touchdown PCR) tm nhit bt mi ti u (khong cch nhit mi chu k thng l 0.5C). 3. i vi cc cp Tm cao hoc t bt cp, c loop th c th khc phc bng PCR 2 bc (95C v 68C). Giai on ko di chui (elongation): Tng hp chui DNA mi ging chui DNA gc. Sau khi mi (primer) tm kim v bt cp b sung vi trnh t ch, nhit ca phn ng li nhanh chng chuyn v nhit thch hp vi hot ng ca enzyme DNA polymerase thng l 720C v gi nhit ny 30 giy n 2 pht ty theo chiu di ca on DNA ch cn tng hp. Trong qu trnh tng hp DNA polymerase s ly nucleotide (A,T,G,C) t mi trng phn ng gn vo u 3 ca mi (primer) theo nguyn tc b sung vi trnh t trn phn t DNA hoc cDNA ch. Nng lng dng trong qu trnh gn kt ny l ATP cng c trong mi trng phn ng. Kt qu qu trnh ny phn ng PCR to ra hai phn t DNA mch i mi t mt phn t DNA mch i ban u.

Nhng ch trong giai on ko di: 1. Nhit ko di thng tin hnh 72C tuy nhin, hot tnh 5'-3' DNA polymerase vn xy ra nhit 68C 2. Thi gian ko di ph thuc hot enzyme DNA polymerase, chiu di sn phm v loi template. 1) i vi DNA ng nht (plasmid, lambda hoc BAC DNA) th tc c th t ti 15s/kb; 2) i vi DNA genome ln, phc tp th tc thng 30s1min/kb.

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Phng php PCR

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Nh vy sau nhiu chu k khuych i (thng t 30-40 chu k) t mt phn t ch ban u phn ng PCR to ra v s phn t bn sao (khoang 106 ban sao) c kch thc xc nh mong mun. Cui cng sn phm PCR c nhum vi Ehididum bromide v em quan st s pht quang di tia UV (bc song 312 nm) sau khi phn tch sn phm PCR trn gel agarose theo kch thc.

S phn ng chui Polymerase

Cc chu k ca k thut PCR

4. Thit k primer (on mi) Vic la chn mt primer c ngha quan trong trong vic ng dng PCR c hiu qu v chnh xc cao. V th ta phi c mt thit k chnh xc v oligonucleotide primer. Trnh t ca primer c chn xc nh kch thc v v tr ca sn phm PCR, cng nh Tm ca vng

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Phng php PCR

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c khuch i. Primer c thit k ng c th gip ta trnh to ra cc sn phm PCR khng dc hiu (non-specific). Mc nh ca vic to ra primer l c c s cn bng gia hai kt qu: s dc hiu v hiu sut khuych i. S c hiu c xem xt trn c s xut hin bao nhiu ln hin tng gn primer nhm (mis-priming) trn tng s ln gn (priming) tc l s lai gia primer v khun mu ti v tr ch ca n. Hiu sut l s tip cn vi l thuyt veefkeets qu sn phm, trong mi chu k PCR, mt cp primer c th khuch i ra mt sn phm. Vi mt trnh t DNA c cung cp ngi ta c th phn tch primer trn computer c kt qu cn bng gia hai mc tiu ni trn. 4.1. Chiu di primer Tnh dc hiu ca sn phm PCR thng ph thuc vo chiu di ca primer v nhit . Cc oligonucleotide c kch thc khong 18 -24 base c xu huwnowngs tr thnh nhng trnh t dc hiu nu nhit ca PCR c nhit ch sai khc v so vi Tm ca primer (theo l thuyt). Primer c chiu di cng ln th kh nng ca n cng nh. Trong qu trnh khuch i , mt s c no trong qu trnh cng lm gim kt qu ca PCR. ti u ha PCR, ngi ta s dng cc primer c kch thc ti thiu sao cho m bo Tm vo khong 54oC hoc hn cht t, iu ny s cung cp nhng c hi tt nht duy trnh tnh c trng ca sn phm PCR v hiu sut phn ng cao. Nhng olignucleotide ngn ( 15 base hoc ngn hn) c s dng hn ch trong quy trnh PCR. Chiu di ti thiu cho hu ht primer l 18 nucleotide v ngi ta thng thit kt primer t 18-24 mer. Primer cng ngn th qu trnh gn primer vi khun mu DNA cng nhanh, to thnh si i n nh trong tng hp DNA. Thng thng cc prime c 28-35 mer cn cho vic khuch i cc trnh t c mc d hp cao. i vi nhng trnh t 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 cc primer di hn cng thc ny c gi tr gn ng. 4.2. Nucleotide cui cng ca primer V tr u 3 ca primer nn c xc nh cn thn, v n 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

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u 3 ca primer. Thng thng, vic thm vo mt trnh t khng lin quan vo u 5 vn khng lm thay i qu trnh gn ni. Nhim v ca u 3 trong primer l kim sot vic gn primer nhm v ngn cn hin tng tng ng trong cng mt primer. Nu khng thn trng trong vic xc nh u 3 th hin tng primer-dimers s x ra, v vi tnh cht b sung cho nhau sn phm ca PCR lc by gi s chnh l s khuch i ca chm primer ch khng phi DNA khun mu. trong trng hp c nhiu primer cng a vo trong cng mt phn ng (multiplex PCR) chng ta cn phi kim tra gp i s b sung cho nhau ca cc primer. 4.3. Hm lng GC v nhit nng chy Tm Primer ca PCR phi duy tr c hm lng GC mc c th c. Nhng oligonucleotide c 20 base vi hm lng 50% GC th c nhit nng chy Tm vo khong 56-62oC s to iu kin qu trnh tt nht. Nu Tm cng ln th hin tng gn primer nhm cng cao. Do n, khi thit k primer cn ch c bit n hm lng GC. 5. Phn tch sn phm PCR bng in di gel agarose in di agarose thng c s dng xc nh sn phm PCR da trn kh nng phn tch cc on DNA c kch thc khc nhau khi sn phm PCR di chuyn qua mang li cc vi l c trong gel agarose. Do phn t DNA tch in m cao. Khi phn t DNA c t di mt in trng xc nh, nhng phn t DNA tch in m ny s di chuyn v pha cc dng ca in trng, trong trng hp in di agarose chng s di chuyn qua gel agarose c t trong dung dch m in di. Cc phn t cng nh c kh nng di chuyn cng nhanh trong gel agarose v pha cc dng so vi cc phn t DNA ln hn. Nguyn tc ny cho php k thut in di phn tch cc on DNA c kch thc khc nhau. Sn phm PCR c trn vi dung dch t mu (loading gel) vo chuyn vo cc ging trn gel agarose thc hin phn tch di tc ng ca in trng. Gel agarose c cha sn Ethidium bromide v th khi DNA di chuyn trong gel agarose s to lin kt vi Ethidium bromide
B sn phm in di

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Dung dch t mu (loading gel) cha cht c t trng nng (nh ng sucrose hoc glycerol) li cun DNA xung y ging trn bn gel agarose v ngn cn khng cho DNA khuch tn vo dung dch m in di. Ngoi ra, trong dung dich t mu cn cha cht ch th tch in m. Cht ch th di chuyn cng vi DNA gip cho d dng c lng s di chuyn ca DNA trong bn gel. Cht ch th thng dng l Xylene cyanol v Bromophenol blue, chng di chuyn cng vi nhng phn t DNA c chiu di tng ng ln lt l 5000 bp and 300 bp m din di: m in di gip n nh cc iu kin mi trng cho php qu trnh di chuyn ca DNA c n nh trong sut qu trnh in di. C mt vi loi m in di thng dng trong phn tch in di agarose: Tris acetate EDTA (TAE), Tris/Borate/EDTA (TBE). Kh nng m ca dung dch m TAE thp hn so vi TBE. Tuy nhin, TAE li cho php phn tch tt i vi nhng on DNA ln hn. iu ny ng ngha vi vic phi dng in th thp hn v mt thi gian in di nhiu hn. Kt thc qu trnh in di bn gel s c t di tc ng ca tia UV, cc on DNA sn phm PCR sau khi phn tch theo kch thc s c hin th di dng bng vch mu vng sng ti nhng v tr khc nhau tng ng vi kch thc chiu di ca chng.

Phn tch sn phm sau khi chp UV

Ethidium bromide c sn trong bn gel s chn vo gia cc baz nit ca phn t DNA dn n chng bm vo phn trung tm ca phn t DNA. S pht sng l do phc hp DNAEthidiun bromide pht ra khi chu tc ng ca tia UV.

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Ghi ch: 1. Khu thao tc tch chit vt liu di truyn, khu pha ch ho cht v khu in di sn phm PCR phi tch bit, trnh trng hp DNA t khu tch chit v khu in di ngoi nhim vo trong ho cht trc khi em phn ng. 2. T thao tc gn n cc tm dng cho pha ch ho cht cho phn ng PCR. 3. Gel agarose chy in di phi c chun b trong mt t ht kh c. 6. Cc ch tiu nh hng n phn ng PCR 6.1 DNA Template (DNA mu) Phn ng khuch i ti u xy ra trn DNA tht tinh sch nhng nhiu k thut chn on bng PCR vn t kt qu tt vi DNA thu nhn trc tip t dch chit t bo. Lng DNA mu s dng cng c khuynh hng gim (1 microgam xung cn 100 ng) vi vic s dng cc polymerase cho hiu qu cao. Hn na, vic gim lng mu ban u cn hn ch c cc khuch i k sinh to nhng sn phm ph khng mong mun. Mt u im ln khc ca phng php PCR l cho php khuch i c nhng mu DNA khng c bo qun tt, b phn hu tng phn nh trong cc vt mu lu ngy, ho thch, tc, mng tay ca ngi cht, Phn ng PCR s khng xy ra c nu bnh phm khng c nucleic acid ch. Mt trng hp khc l trong bnh phm c nucleic acid ch, nhng do bnh phm c sa son khng thch hp hoc khng ng cch nn nucleic acid ch khng bc l ra; hoc trong bnh phm hy cn cc cht c ch phn ng PCR. Trong nhng trng hp ny kt qu PCR s m tnh, nhng l m tnh gi. V vy vn sa son bnh phm cho th nghim PCR phi c coi trng, c bit trong cc phng th nghim p dng PCR chn on pht hin bnh. Nng ti u DNA template l t: 1) i vi cc mu DNA tng i ng nht (plasmid, lambda hoc BAC DNA): 1 pg - 10 ng / 50 l tng th tch phn ng. 2) i vi mu DNA genome th t 50 - 500 ng/50 l tng th tch. 6.2 Enzyme polymerase Enzyme c s dng u tin l on Klenow ca DNA polymerase I. V y l enzyme khng chu nhit nn thao tc phc tp v hiu qu thp (phi thm enzyme mi vo phn ng sau mi ln bin tnh v enzyme c b nhit phn hu, nhit lai thp khin s

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khuch i k sinh rt cao,). Phng php PCR chuyn sang mt bc ngoc mi cng vi s pht hin mt DNA polymerase chu nhit c tch chit t mt vi khun sui nc nng, Thermus aquaticus. Ezyme ny- Taq polymerase- khng b ph hu nhit bin tnh v xc tc s tng hp t u n cui qu trnh phn ng. Ngy nay, nhiu polymerase c a ra th trng vi nhiu chc nng chuyn bit hay hon thin hn. Tth polymerase, mt enzyme tch chit t Thermus thermophilus, c kh nng hot ng nh mt enzyme phin m ngc khi c mt RNA khun v ion Mg 2+; nhng vi s hin din ca DNA khun v ion Mg2+, Tth polymerase li xc tc phn ng khuch i DNA. Enzyme ny cho php khuch i bn mu l RNA thng qua s hnh thnh cDNA. Khi s dng Ezyme polymerase cn ch mt s im: 1. Cc c tnh enzyme DNA polymerase v nh ngha n v (unit) ca mi loi (hng) sn xut l khc nhau. Cn kim tra thng tin trn cc t hng dn i km ha cht. 2. Lng enzyme cn thit l ph thuc vo lng DNA template v kch thc phn ng PCR. Tuy nhin, thnh phn ny ch nn hiu chnh khi 1) sn phm PCR ln hn 5kb; 2) c kh nng hot tnh ca enzyme b gim st do thiu st khu bo qun; 3) tin hnh hiu chnh bng nhiu cch nhng sn phm PCR vn thp hoc khng c g. 3. Khng nn tng enzyme qu 2U (5l) trn 50 l tng th tch phn ng 4. Vic thay i loi DNA polymerase c th lm thay i hiu sut ca phn ng PCR. 5. Khi tnh ton chin lc cloning bng h thng TA vector, cn lu DNA polymerase c c tnh to u treo A u 3' hay khng. Nu khng th cn phn ng b sung s dng Klenow v ATP. 6. Tnh chnh xc ca enzyme DNA polymerase ph thuc vo hot tnh 35 exonuclease. Ty thuc mc ch th nghim m cn la chn loi DNA polymerase c hay khng c hot tnh ny. 6.3 Primer (on mi) on mi tc l nhng on DNA n (oligonucleotide) c kch thc ch vi chc base (18-30), c th bt cp theo nguyn tc b sung vo on khi u v on kt thc ca chui DNA ch khi chui ch ny c bin tnh thnh si n. Trong th nghim PCR, on mi c hai vai tr chnh : (1) Quyt nh nn tnh c hiu ca th nghim, v nu on mi c chn cng c hiu cho chui ch, ngha l ch c th bt cp trn chui ch m khng th bt cp c trn cc chui DNA khc ngoi chui ch, th sn phm PCR cng c hiu v th nghim PCR cng c hiu. (2) Khi ng men polymerase v men polymerase ch c th bt u tng hp si b sung cho chui DNA ch mt khi n nhn dng c u 3', (l u m n xc tc cho mt dNTP c gn vo) ang tnh trng si i. Thng thng trong phn ng PCR, ngi ta dng mt cp mi, gi l primer set, trong c mt mi ln gi l

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up-stream primer v mt mi xung gi l down-stream prime. Cp mi ny quyt nh nn kch thc ca sn phm PCR. Chng cng bt cp trn chui ch xa nhau bao nhiu th kch thc ca sn phm PCR cng ln by nhiu v ngc li, cng gn nhau bao nhiu th kch thc cng nh by nhiu. Vic chn mi l giai on quyt nh ca phng php PCR, v phi tun th mt s nguyn tc: + Trnh t ca mi c chn sao cho khng th c s bt cp b sung gia mi xui v mi ngc, v cng khng c nhng cu trc kp tc do s bt cp b sung gia cc phn khc nhau ca mt mi. + Chn trnh t mi c khong 50% GC. Trnh G v C u 3 ca mi v n c th lm tng c hi to ra hin tng primer-dimers (hai mi bt cp vi nhau). Trnh chn cc vng c trnh t tng ng hn ch cc mi t gn vi nhau. + Tnh ton nhit nng chy (Tm) ca mi vi tng s 4oC cho GC v 2oC cho AT, sau tr i 5oC t gi tr ny v y chnh l nhit (Ta) ca mi. Nhit phi c gi tr thp hn nhit nng chy ca mi. s khc nhau t 4 6oC gia Tm ca mi v Ta khng nh hng n hiu sut ca PCR. + Nhit gn mi (Tm) ca mi xui v mi ngc khng cch bit qu xa. Thnh phn nucleotide ca cc mi cn bng trnh cc cp GC lp i lp li nhiu ln. + Cc mi chn phi c trng cho trnh t DNA cn khuch i, khng trng vi cc trnh t lp li trn gen. + Trnh t nm gia hai mi xui v ngc khng qu ln, phn ng PCR s ti u trn nhng trnh t nh hn 1kb 6.4 dNTP (deoxy Nucleoside Triphosphate) L nguyn liu tham gia to nn mch DNA mi, gm bn loi dATP, dTTP, dGTP. C cu to gm mt ng deoxyribose c gn mt base, c th l adenine (dATP) hay thymine (dTTP) hay Cytosine (dCTP) hay guanine (dGTP) , carbone s 1 (C1). Ba phn t phosphate (triphosphate) c gn ti carbone s 5 (C5) ca phn t deoxyribose ny v y chnh l ni m dNTP gn vo u 3' ca chui b sung trn chui ch. Nng lng cho phn ng ny xy ra c ly t cc ni phosphate giu nng lng ca triphosphate trn dNTP. cng chnh l l do ti sao phi l dNTP chng khng phi l dNDP (diphosphate) hay dNMP(monophosphate). Khi s dng dNTP cn ch : + dNTPs l loi ha cht km bn cn lu khi s dng v bo qun. Nn chia nh lng dNTPs gim s ln lm tan, s dng li.

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+ Lng dNTP ti u l 200 M of mi loi dNTP. Vic tng dNTP khng lm tng ng k lng sn phm PCR so vi vic hiu chnh cc yu t khc. 6.5 Buffers (dung dch m) Thng cha mui m Tris HCl 10 mM, KCl 50Mm v MgCl2 21.5mM. Ngoi ra dung dch m PCR cn c th cha 0.001% BSA hay Gelatine v trong mt s phn ng PCR cn c th thm tween hay formamide na. Trong cc thnh phn trn, c l nh hng ln th nghim PCR nhiu nht l nng MgCl2, v vy c c mt th nghim PCR c nhy cao, ph ng r nt, ngi ta phi ti u ha phn ng bng cch thm d mt nng MgCl2 thch hp nht. + Vic tng nng ion Mg2+ c th lm xut hin thm cc sn phm khng c hiu. Tuy nhin nu thiu ion Mg2+ th dn n lng sn phm PCR thp. + Nng ion Mg2+ ti u ph thuc vo loi DNA polymerase, loi DNA template v thnh phn buffer (mt s buffer thng mi c sn 1 lng nht nh ion Mg2+). + D tm nng Mg2+ ti u thng thng bng gradien nng t 0.5 n 2 mM vi cc khong cch mi bc l 0.2 mM. Hiu sut, tnh chnh xc ca enzyme DNA polymerase ph thuc nhiu vo h buffer s dng. Thng thng nn s dng ng loi buffer tng ng vi enzyme do nh sn xut khuyn co. Mt s buffer cha cht hot ng b mt c th nh hng n cc phn ng microarray hoc DHPLC. 6.6 Mt s yu t khc 6.6.1 S lng chu k ca phn ng PCR Trong thc t, ngi ta khng vt qu 40 chu k cho mt phn ng PCR v phn ng PCR din tin qua hai giai on, trong giai on u s lng bn sao tng theo cp s nhn t l vi lng mu ban u, sau hiu qu khuch i gim hn v nhng nguyn nhn sau: + S phn hy v cn kit cc thnh phn ca phn ng + S xut hin cc sn phm ph c ch phn ng + Cc bn sao va c tng hp khng c kt hp vi mi m bt cp vi nhau S chu k cho mt phn ng ty thuc s lng bn mu ban u. V d: nu s lng bn mu l 105 th cn 25-30 chu k, nu s lng bn mu l 102-103 th s chu k l 35-40 6.6.2 Thit b v dng c cho phn ng PCR + Thit b dng tin hnh phn ng PCR ch cn p ng c yu cu thay i nhit tht nhanh v chnh xc. + Mi th nghim ca mt nghin cu cn c tin hnh trn cng mt loi thit b. Thc hin: Phm Huy Quang Trang 15

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+ ng nghim dng cho cc phn ng cu cng mt nghin cu phi thuc cng mt kiu v c tnh truyn nhit ca cc ng ny cng nh tip xc gia ng v b phn to nhit ca thit b c nh hng ln n qu trnh khuych i. 7. Gii php ti u ha cho phn ng PCR 7.1. Khng c sn phm hoc lng sn phm qu thp + Lp li th nghim phng li thao tc. + S dng ng dNTP mi, c cht lng m bo. + Tng lng DNA template, in di kim tra li nng DNA template. + Tinh sch DNA template bng chit phenol, ta cn hoc kit tinh sch hoc thi gel. Nu c th cn tin hnh tch chit mi DNA template bng cc phng php tch chit khc nhau. + Tng thi gian ko di i vi cc sn phm PCR trn 0,5 kb. + Tng s chu k PCR ln 35, 40. + H thp nhit bt mi xung 1 n 2C. Hoc s dng touchdown PCR. + Dng PCR nh bt vi nhit bt mi kh thp (~45C) trong 10 chu k u v nng cao nhit n Tm thp nht 20 chu k tip theo. + Tng nhit bin tnh (ti a 98C) v iu chnh thi gian bin tnh DNA i vi cc mu DNA ca genome ln hoc khi khuych i cc on lp SSR. + Kim tra cc iu kin ca primer, thit k cp primer mi chy nestPCR. + Tng nng enzyme DNA polymerase + Thay i loi enzyme DNA polymerase v h buffer 7.2. Vt smear kch thc ln hn sn phm mong mun + Gim nng enzyme DNA polymerase + Rt ngn thi gian ko di + Gim s chu k nhit PCR xung ti a 25. + Tng nhit bt mi ln 2C hoc th vi PCR 2 giai on hoc nestPCR + Thay i nhit bin tnh. + Th chy gradien nng Mg2+ + H thp nng primer. 7.3. Bng sn phm ph r nt vi kch thc thp hn sn phm mong mun + Tng nhit bt mi 2C v/hoc tng thi gian bt mi + Tng thi gian ko di thm 30s. + Gim nng DNA polymerase. Thc hin: Phm Huy Quang Trang 16

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+ Tinh sch DNA template bng thi gel. Hoc thy phn DNA bng mt s RE trc khi dng lm template (hiu qu khi bng sn phm ph kch thc kh ln >= 1kb). + Gim nng primer. + Thit k cp primer mi. 8. Cc u im v hn ch ca k thut PCR 8.1. u im: - Thi gian thc hin cc nhanh: ch cn 3 gi khuch i mt trnh t DNA c quan tm. So vi phng phap tao dong cua ky thuat tai to hp DNA
phai mat ca tuan hoac lau hn.

- n gin v t tn km: c thc hin trong ng nghim plastic nh gm cc thnh phn ti thiu c s dng ng thi. Trong khi o, phng phap tao dong ien hnh
can cac vat lieu at tien nh mang, nucleotide triphosphate mang dau phong xa va viec thc hien can cac thao tac kheo leo ac biet.

- tinh sch ca mu khng cn cao: PCR c th thc hin vi cc mu nucleic acid th. V
du, mau mau hay cac dau vet trong phan tch phap y. ieu nay ngc vi ky thuat tai to hp DNA, oan gen hoac vector eu can tng oi tinh khiet - Co the phat hien nhng vi khuan kho nuoi cay. Viec tang sinh la n gian hoac khong can thiet. - Hoa chat dung cho phan ng PCR de tm va de ton ch hn so vi trng hp huyet thanh hoc. Khong can dung cu va moi trng chuan oan phc tap, co the thc hien hien trng. - t ton kem ve mat nhan s, co the t ong hoa e lam giam chi ph phat hien vi sinh vat gay benh

8.2. Hn ch: - Kch thc ca trnh t cn khuch i: phng php PCR khng hot ng c vi nhng on DNA ln hn 3kb. Vic s dng PCR i vi cc di di 1.5kb cho kt qu tt. Vi nhng di ln hn, iu kin ti u cho phn ng phi c xc nh qua thc nghim. - S ngoi nhim: ngun ngoi nhim ln nht thng l cc sn phm khuch i ca nhng ln thao tc trc. Ngi ta chng minh c rng m np cc ng nghim sau mi ln khuch i trong mt khong khng gian kn nh phng th nghim s khin cho cc phn t

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c khuch i thot ra khi ng nghim bay l lng trong khng kh v bm vo tng, ca, thit b, dng c ri nhim vo cc phn ng tin hnh sau . C th khc phc vn ny bng mt s bin php sau: + Cc cng on thao tc khc nhau nh thit lp phn ng PCR v phn tch cc sn phm khuch i phi c tin hnh nhng a im cch xa nhau. + Dng c dng thit lp phn ng (micropipette) khng s dng vo cc thao tc khc. u tp s dng vi micropipette phi c lp lc trnh s nhim u micropipette bi cc phn t khuch i khi ht dung dch phn ng. + Dng tia t ngoi loi b cc phn t cn li t cc ln khuch i trc. + Tt c mi thnh phn ca phn ng u c chia thnh nhng lng nh, tnh ton sao cho 1, 2 ln thao tc. + Ngoi ra, cc hng sn xut cn tung ra th trng nhiu h thng cho php loi b hon ton s ngoi nhim. V d hng Perkin-Elmer-Cetus xut vic s dng dUTP thay v dTTP; vic thay th ny khng nh hng n hn ng. trc mi ln phn ng k tip, ngi ta cho thm vo dung dch phn ng uracyglycosylase; enzyme ny s phn hy tt c cc DNA c mang dUTP nhim t ln trc. ng thi, enzyme s b phn hy bi nhit ngay t ln bin tnh u tin. Bt li ca cc h thng ny l gi thnh cao. - Cc sai st gy ra do Taq polymerase: s sao chp bi Taq polymerase cho t l sai kh cao (10-4, ngha l c 1000 nucleotide th enzyme gn sai 1 nucleotide). Ta khng th loi b hon ton cc sai st ny m ch c th gim bt; v d nh m bo s cn bng nng cc nucleotide trong phn ng, xc nh trnh t ca nhiu sn phm khuch i t nhiu thao tc ring bit, so snh trc khi i n trnh t chnh thc - S c che hoat tnh cua Taq DNA polymerase bi thanh phan cua mau vat, do mau thng co nhng thanh phan phc tap. Tuy nhien, viec tach chiet va tinh che DNA t thc pham hay moi trng trc khi thc hien phan ng PCR thng cho phep loai bo nhng hp chat c che. - Mat o vi sinh vat hien dien trong mau thc pham thng thap, nen a so trng hp can co bc nuoi cay lam giau e co c mat o vi sinh vat u cho viec phat hien bang PCR. - Phng phap nay khong phan biet c te bao song vi te bao chet, do vay co the dan en trng hp dng tnh gia do DNA t te bao chet.

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- Phng phap nay ch phat hien c vi sinh vat co trong mau ch khong xac nh c so lng vi sinh vat co trong mau. - Chi ph cho viec phat hien cac vi sinh vat n le trong thc pham thng ton kem. Do vay, e giam chi ph ngi ta thng s dung nhieu cap moi trong mot phan ng PCR (goi la phan ng multiplex PCR) e co the phat hien ong thi nhieu vi sinh vat gay benh khac nhau. 9. Mt s ng dng ca PCR PCR c phm vi ng dng rt rng trong cng ngh sinh hc hin i.Theo nguyn tc trn, c nhiu b sung v ci tin dng PCR cho nhiu mc tiu khc nhau. y ch gii thiu mt s ng dng chnh. Trong nghin cu genome + Nhn bn phn t bng PCR + PCR ti t hp + K thut footprintinh DNase I + sang lc th vin gt11 +Multiplex PCR Trong y hc +Pht hin t bo T/vius gy bnh ung th mu + Pht hin vius vin gan B Pht hin vius Dengue gy bnh st xut huyt + Pht hin vi khun lao 9.1. S dng PCR to nhanh cc gen tng hp S dng phng php PCR hai giai on (two-step PCR) to nhanh cc gen tng hp. Phng php ny c xy dng trn c s nhng nucleotide chng ln nhau (overlapping), do c th c s dng to ra on DNA tng hp thng qua nhiu vng khuch i PCR. Kh nng to ra DNA tng hp c nhiu ng dng ty thuc vo thit l ca chng ta. Ngi ta c th thit k mt gen tng hp c cha codon c dung biu hin protein trong c th sinh vt. Phng php ny c hon tt nh s gii m ngc chui aminoaxit ca protenin mong mun. thay i cu trc codon, gen tng hp c th c thit k c nhng v tr ct hn ch thun li phc v cho th nghim to dng v sau. Nguyn tc ca PCR hai giai on l hai phn ng PCR xy ra lin nhau, phn ng th nht ca PCR pht sinh ra si khun mu tng ng vi gen tng hp v sau n c

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khuch i trong PCR ln th hai (hnh 1). Trc khi bt u qu trnh ny, ngi ta phi thit k cu trc v xc nh trnh t nucleotide ca gen tng hp m mnh mong mun. Sau , trnh t cc oligonucleotide (primers) theo chiu di ca gen phi c thit k v tng hp. Thng thng, ngi ta tng hp cc oligonucleotide theo s chn vi chiu di khong 16-30 nucleotide. 9.2 To dng cDNA bng PCR. K thut to dng kinh in cDNA i hi nhiu cng sc v xy dng th vin bo qun Bcteriophage hoc plasmid, n cn mt khi lng cn, vic chn lc mt s lng ln cc phage hoc plasmid c tnh cht ti t hp. C ba hn ch trong phng php ny: - Cn c mt lng c cht (t nht 1g) mRNA tinh sch lm vt liu khi u xy dng th vin vi mc a dng y cho nghin cu. - Kh khn thuc v bn cht bn trong ca nhng phn ng enzyme tip ni nhau lm cho phn ng to dng cDNA c kt qu thp v nhng dng gen b t on. Vic sng lc mt th vin vi k thut lai i hi nhiu thi gian. K thut PCR hin nay c th gip chng ta khc phc c nhng nhc im ni trn, lm cho vic to dng cDNA tr nn d dng hn. S dng hai primer c hiu ca gen, mt cDNA c trnh t bit r, ngi ta cho khuch i trong PCR. Tuy nhin, ci kh l lm sao phn lp c nhng bn sao ca cDNA hon chnh t mRNA, trn c s thng tin v trnh t rt hn ch. Trnh t cha bit ny nm k mt on nh ca trnh t bit r. Trnh t cha bit khng th khuych i bng phng php PCR thng thng. Do , phng php PCR m neo v phng php PCR o ngc c pht trin trong thi gian gn y gii quyt vn ny.

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Hnh 1: 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.

9.2.1. PCR m neo K thut PCR m neo c mt im chung l: to dng DNA i t mt on trnh t bit ri ti on trnh t k cn cha bit nh s gip ca mt primer c hiu i vi gen ti mt u si n v mt primer tng hp ti mt u si n khc. Do ch c primer c hiu i vi gen mi c th neo trong PCR lm d dng hn cho vic thu thp mt lng ln sn phm khuch i khng c hiu so vi PCR chun. K thut ny khc vi PCR chun l ch cn mt primer c hiu cho phn ng. Trong k thut PCR m neo, mt ui nhn to ca cc gc dA c them vo on cui ca DNA khun mu. S khuch i DNA xy ra khi c primer ca mt trnh t bit trc v mt primer th hai c cc gc oligo (dT). 9.2.2. PCR o ngc. Phng php ny c thun li ln hn nh khuch i trnh t k cn cha bit bng hai primer c hiu ca gen. PCR o ngc hot ng theo nguyn tc to dng si n mt trnh t DNA. Sau , DNA ny c ct bng RE v tr tng ng to DNA ch, tip theo n hot ng theo chu trnh c tnh cht monomer.DNA ny c khuch i ln bng cc primer tng ng ti hai u si n ca DNA c trnh t bit.

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Hnh 2:

ng dng PCR o ngc

Ban u l si 5-3 ca mRNA cho vo phn ng phin m ngc thnh si n cDNA 3-5, tip tc tng hp si n ca cDNA ln th hai, cho ra si i cDNA (5-3 v 3-5). Si ny c cha mt trnh t bit r. K thut to dng lm cho cDNA thng bin thnh cDNA vng si i, tip n, m vng nh RE ct trnh t c bit lm i, mt na u si thng bn ny, mt na bn kia. Ko thng si DNA v khuch i trong PCR (hnh 2). 9.3. ng dng trong di truyn PCR gip c lc cho vic lp bn gen 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 qu 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 sn phm PCR ca nhiu ging c c im khc nhau trog cng loi, vi nhiu primer khc nhau c th gip xc nh bn gen ca nhiu sinh vt. Hin nay, ngoi RAPD cn c nhiu loi ch thi phn t (molecular make) khc c pht trin nh a hnh chiu di cc on ct hn ch (restriction fragment length polymorphism RELF), v v tinh (microsatellite) hay cn gi l s lp li cc on nucleotide n gin (simple sequencerepeats SSR), v a hnh chiu di on DNA c khuch i 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 a dng di truyn (generic diversity), nh v gene (gene location) v lp bn

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gen do chng n gin v mt k thut, tit kim thi gian v c hiu qu cao. PCR hin nay cn c dung thay th cho in di esozyme phn loi thc vt, xc nh mi quan h gia chng mt cch chnh xc. 9.4. 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 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.

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phng php ny c th khuch i mt on RNA bng phng php RT-PCR: 1. 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. 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. 2. Real-time PCR 2.1. 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

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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. 2.2. 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). 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).

Hnh 3: ng cong khuch i. Tn hiu hunh quang trnh by tr ng nn. u tin, tn hiu hunh quang duy tr mc nn (background), v vic tng tn hiu

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hunh quang l khng th pht hin c (cc chu k 1-18, Hnh 3) 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. 2.3. 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).

PHN III: KT LUN

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Do cc ng dng cc k to ln v k diu trong mi lnh vc, PCR tht s lm c mt cuc i cch mng trong sinh hc phn t trong thi im hin nay. Vi k thut v cng ngh ngy cng tin b, cc thuc th ngy cng r hn v tt hn, gi my chu k nhit ngy cng h v c bit l vic s dng h thng ni ti chng ngoi nhim, th nghim PCR khng cn l mt th nghim qu cao cp ch thc hin c ti cc phng th nghim hin i. C th ni PCR hin nay hon ton c th trin khai ti cc phng th nghim trung bnh ti cc nc ang pht trin nh chng ta. Ngy nay, bn cnh phng php PCR truyn thng, ngi ta pht trin thm rt nhiu ci tin cng nh k thut mi da trn nn tng PCR c th p dng cho nhiu loi i tng mang vt cht di truyn khc nhau, nhng tin b ny c p dng vo trong thc tin s gip chng ta c th pht hin v c nhng phn ng nhanh nhy kp thi trc tnh hnh din bin phc tp ca cc loi bnh dch nguy him trn th gii hin nay nh H1N1, H5N1, Sars, cng nh gp phn pht hin iu tr nhng bnh v ang e da th gii nh Lao, Vim gan, . Vi ch trng i tt n u, nghin cu, p dng v trin khai cc tin b khoa hc vo nng cao cht lng i sng ca nhn dn, Vit nam p dng kh rng ri phng php PCR v cc k thut tin tin khc trong nghin cu Sinh hc phn t, v bc u thu c mt s kt qu ng ghi nhn, tin rng trong tng lai chng ta s cn gt hi c nhiu thnh cng hn na trong s nghip nghin cu khoa hc ca nc nh.

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1. H Hunh Thu Dng, Sinh hc phn t. NXB Gio Dc, 1997. 2. Phm Thnh H. Di truyn hc. NXB Gio Dc, 2007, 3. Nguyn Th Thanh, Bi ging Sinh hc phn t, i hc ty nguyn, 2008. 4. Nguyn Hong Lc, Cng ngh DNA ti t hp, NXB i hc quc gia TP HCM, 2007 5. http://www.sinhviennonglam.com/forum/showthread.php?t=3114 6. http://magicbmt.com/forum/f82/ky-thuat-pcr-can-ban-16839/ 7. http://www.semvn.net/RC/modules.php?name=Forums&file=viewtopic&t=2 8.http://tusach.thuvienkhoahoc.com/wiki/S%C3%A1ch:C%E1%BA %A9m_nang_SHPT/Ch%C6%B0%C6%A1ng_18._K%E1%BB%B9_thu %E1%BA%ADt_PCR_c%C4%83n_b%E1%BA%A3n 9. http://hau1.info/forum/showthread.php?t=6609 10. http://www.khoahocthuysan.org/forums/viewtopic.php?f=6&t=36 11. http://lop06sh1d.forumup.com/about41-lop06sh1d.html 12. http://www.bme.vn/bmevn/bme/lien-nganh/ly-hoa/28-virus-h5n1-va-phng-phap-pcr

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