H tn: inh Hu Hiu MSV: 1153060841

BI THO LUN
Cc phng php nghin cu, phn tch carbohydrate, lipit v nucleic acid
I. Cacbohydrate:
1. Lch s nghin cu: Sir Walter Norman Haworth (19.3.1883 ti Chorley, Lancashire 19.3.1950 ti Barnt Green, Worcestershire) l mt nh ha hc ngi Anh, ni ting v cng trnh nghin cu axt ascorbic (vitamin C) khi ng lm vic i hc Birmingham. ng ot gii Nobel Ha hc nm 1937 "cho cng trnh nghin cu v cc carbohydrate v vitamin C". Gii ny c thng chung vi Paul Karrer, nh ha hc ngi Thy S v cng trnh nghin cu cc vitamin khc ca ng ta. ng quyt nh theo hc ha hc i hc Manchester nm 1903, sau khi lm vic mt thi gian trong nh my ch to linoleum ca ngi cha. ng ly quyt nh ny, mc d c s khng ng tnh ca cha m. Sau khi tt nghip cp thc s khi hc vi William Henry Perkin, Jr., ng tip tc hc i hc Gttingen vi Otto Wallach v u bng tin s. Nm 1912 Haworth lm ging vin United College ca i hc St Andrews ti Scotland v quan tm ti ha hc carbohydrate (carbohydrate chemistry), do Thomas Purdie (1843-1916) v James Irvine (1877-1952) nghin cu i hc St Andrews. Haworth bt u nghin cu trn ng nm 1915 v pht trin mt phng php mi ch to cc methyl ether ca ng, dng methyl sulfate v alkali (nay gi l Haworth methylation). Sau ng bt u nghin cu v mt cu trc ca cc disacarit. Haworth t chc cc phng th nghim i hc St Andrews sn xut cc ha cht v dc phm cho chnh ph Anh trong Th chin th nht (1914-1918). ng c b nhim lm gio s khoa ha hc hu c i hc Durham nm 1920. Ba nm sau, ng lm gio s ha hc i hc Birmingham. Nm 1934, lm vic chung vi Sir Edmund Hirst, mt nh ha hc Anh khc, ng c th tng hp c vitamin C. ng pht trin mt phng php n gin biu th trn giy cu trc 3 chiu ca ng. Cch trnh by ny, nay gi l Haworth projection (hnh chiu Haworth), vn cn c s dng rng ri trong ngnh ha sinh.

2. Phng php phn tch: a. Cc phng php ha hc: Phng php nh lng ng tng s ha tan: c s dng rng ri xc nh carbohydrate tng trong thc phm Carbohydrate c chuyn thnh dng nh lng ng tng sha tan 24 chuyn thnh dng monosaccharide di tc nhn acid v nhit Dng dehydrate cua Carbohydratee s to thnh cc dn xut (furan) c th kt hp vi nhau hoc vi phenol, orcinol, anthrone... S dng Thuc th Phenol (AOAC 988.12) - 1ml dd ng (10-70g ng) + 1ml dd Phenol 5%, trn u + 5ml H2SO4(cn trng), yn 10 pht ri trn u. - un cch thy 2 pht 100C, ngui trong 30 pht. - o mu 485nm. Sdng Thuc th Orcinol 1ml dd ng (10-70g ng) + 2ml thuc th orcinol (2% trong H2SO430%) + 15 ml H2SO4 60%, trn u. un cch thy 80C trong 20 pht. Lm lnh bng nc . o mu 470nm hoc 520nm. Phng php nh lng ng kh: nh lng bng 3,5- Dinitrosalisylic acid nh lng bng Ferricyanur inh lng theo Schaffer Harmann nh lng theo Somogyi-Nelson nh lng theo Bertrand...

Phn tch mono v oligosaccharide: Phn tch nh lng-Sc k bn mng (TLC) Pha tnh l silica gel nh lng bng phng php vt l

 Sc k kh (GC): Yu cu: hp cht phi d bay hi ng to ether vi Trimethylsilane

Sc k lng cao p (HPLC): Stationary Phase: Waters Associates. 840380 Solvent: H2O/CH3CN

 Phn tch tinh bt: nh lng bng cch thy phn ha hc hoc bng enzyme thnh cc n phn t, sau nhn dng phn tch Ch yu l phn tch nh tnh 1. Amylose + IMu xanh Sdng quang phk o mu ca c trng ca amylose-iod 2. Amylopectin + IMu ta

b. Phng php phn tch vt l: S dng cc thit b nh: T trng k Khc x k Phn cc k

II. Nucleic acid:

1. Lch s nghin cu: Nucleic acid l nhng hp cht cao phn t ng vai tr ht sc quan trng trong hot ng sng ca mi c th sinh vt. Chng tham gia vo cc qu trnh cbn ca ssng nh sinh tng hp protein, sinh trng, sinh sn v di truyn. Trong mt thi gian di cc nh ha hc v cc nh nghin cu v sinh l dinh dng coi protein, lipid v carbonhydrate l ba cht quan trng nht to nn c th sng. Quan im cho rng nucleic acid l nhng cu t tr ca nhn v t bo cht mi mi lng qun t khi cht th t ny nucleic acid c chng minh l cht quan trng hn so vi cc cht trc . Nm 1869, ln u tin nh ha sinh hc tr tui ngi Thy S Friedrich Miescher (1844 -1895) pht hin nucleic acid trong nhn t bo. ng t tn l nuclein v nhn thy n tn ti trong nhn t bo (nucleus). i tng nghin cu ca Miescher l nhng bch cu (lymphocytes). Khi dng acid kt ta dch chit xut t nhn ca t bo m ly tbng bng b i (bng cch dng enzyme phn hy protein ca dch d dy l pepsin tiu ha cc phn khc giu protein ca t bo) ng v cng ngc nhin nhn thy rng, nhn t bo cha mt cht khng phi m, khng phi carbonhydrate, cng khng phi protein. N cng khng ging mt cht sng no bit v cha phosphor v nit, ha tan th cho tnh acid. Tnucleic acid l do Altman ngh nm 1889 (ng pht hin ra rng i tng thun tin tt nht chit rt cht nuclein l nhng u tinh trng ca c hi). V thc cht mi u tinh trng ca c hi hon ton tng ng vi mt nhn tbo. iu ch ch phm nucleic acid, Miescher ha tan phn u ca tinh trng (t bo sinh dc c) trong dung dch mui nng cao, sau bng cch thm nc vo gy ra kt ta nucleic acid dng si. Cn phi gi ch phm lnh do trong cc ngy ng thng gi ca ma ng, ng lm vic trong phng khng si.

Thc t, lch s nghin cu ha hc ca nucleic acid gn lin vi tn tui ca Felix Hoppe - Seiler (1825-1895), nh sinh l hc v ho hc rt ni ting ngi c v chnh phng th nghim ca ng Tbingen. Miescher lm vic vi danh ngha l ngi hc tr v cng tc vin khoa hc tr. Mc d Miescher l mt cng tc vin v cng cn thn v y trin vng song Hoppe - Seiler vn thy cn thit phi ch thn lp li th nghim xem c ng l mt cht mi hay khng. Kt qu thc nghim khng nhng khng nh thnh tu ca Miescher m cn thu c thm nhiu dn liu mi rt c trng. Hoppe - Seiler cn pht hin trong nhn t bo nm men cng c cht nuclein ging nh t bo bch cu. Ngi k tc pht trin cng trnh ca Miescher l nh ha sinh hc (ha hc hu c) ngi c Albrecht Kossel (1853 - 1927). Nm 1882, ng phn tch nucleic acid ra nhng phn nh cha acid phosphoric, ng v base cha nit(Kossel tch c hai pyrimidine v t tn l cytosine v thymine, cng nh hai purine vi tn adenine v guanine). Do cng trnh ny ng t gii Nobel vy v sinh l hc vo nm 1910. Mt pyrimidine khc l uracil cng c pht hin sau ny. Trong nhng nm u ca th k XX (1900 - 1932), nh ha sinh hc ngi M gc Nga Phoebus Aaron Theodore Levene (1869 - 1940) xc nh c n v cu to ca nucleic acid l cc nucleotide (hay mononucleotide) v phn bit c hai loi nucleic acid: deoxyribonucleic acid(DNA) v ribonucleic acid (RNA). Nh vy, cho n nm 30 ca th k XX ny, ngi ta bit c thnh phn ca cc nucleic acid. Levene on trc 4 nucleotide khc nhau lin quan n RNA l adenylic acid (cha adenine), guanilic acid (cha guanine), cytidilic acid (cha cytosine) v uridic acid (cha uracil). ng cng githit 4 nucleotide tip theo lin quan n DNA l deoxyadenilic acid, deoxyguanilic acid, deoxythymidilic acid (cha thymidine) v deoxycytidilic acid (cha cytosine). Do cc gi thit ny ca Levene tra ph hp vi hiu bit ca ha hc cho nn chng sm c cc nh ha hc chp nhn. Nhng cho n thi k u nhng nm 1950, chng vn cha c chng minh r rng khi nh ha sinh hc ngi Anh Alexander Robertus Todd (1907-1997) cha tng hp c cc nucleotide ph hp mt cch chnh xc vi cc cng thc ca Levene. Todd xc nhn c rng cc cht ny thc t ging cc hp cht thu nhn c tnucleic acid. Vi cng trnh ny, Todd c trao gii thng Nobel v ha hc nm 1957. Trong nhng nm 1949 - 1953 nh sinh hc ngi M gc o Erwin Chargaff (1905 - 2002) khi phn tch DNA pht hin s cn bng ca cc base trong DNA: snhm purine bng s nhm pyrimidine (purine/pyrimidine = 1), s nhm adenine bng s nhm thymine (adenine/thymine = 1), snhm guanine bng s nhm cytosine (guanine/cytosine = 1). V sau ny ngi ta t quy tc mang tn ng vtng lng cc loi nucleotide cu to nn cc DNA. Trong nhng nm u thp nin 1950, hai nh vt l hc ngi Anh, Maurice Hugh Frederick Wilkins (1916 - 2004) cng vi Rosalind Franklin (1920 - 1958) bt u tin hnh cc nghin cu u tin vcu trc DNA. Wilkins l con trai ca mt bc sngi New Zealand chuyn hc vt l v vo thi k u cuc Chin tranh Thgii thhai, ng lm vic cho spht trin bom nguyn t. Nhng sau cuc Thchin ny, ng dng tt c sc lc ca mnh cho cng vic nghin cu DNA v tr thnh mt trong nhng nh bc hc tin phong trong lnh vc ny nn c coi l nh l sinh hc. Wilkins v Franklin p dng phng php nhiu x Rnghen(X-ray diffraction) vo vic nghin

cu cu trc tinh thca DNA (ly t tuyn c b, thymus). Vi nhng bc nh chp cu trc tinh th DNA c dng chthp, hai tc gi ny gi rng DNA c th gm hai hoc ba mch n bn xon vi nhau. Nm 1953, tcc nghin cu ca mnh kt hp vi cc kt qu nghin cu trc ca Chargaff v Wilkins, hai nh khoa hc trng i hc Tng hp Cambridge l Francis Harry Compton Crick (England; 1916-2004) v James Dewey Watson (USA; 1928-2003) xy dng thnh cng m hnh chui xon kp ca phn tDNA. Vi cng trnh ny, Watson v Crick c trao gii thng Nobel vy hc v sinh l hc nm 1962. T Crick xy dng mt chui xon kp DNA bng ng trong vn nh ng trung tm Cambridge, sau vit: Ngi ta hi ti l khi no th s m vng. Cng vi vic nghin cu cu trc DNA, cc nh khoa hc cng n lc tm hiu vai tr v cc chc nng sinh hc ca n. Vo nm 1944, ngha l sau 75 nm ktkhi pht hin DNA trong nhn tbo nm 1869, nh vi khun hc ngi M OswwaldTheodore Avery (1877 - 1955) chng minh DNA l cht liu di truyn khi bsung DNA chit xut tchng c (chng khng c ca phcu khun Diplococcus pneumoniae bin thnh chng c gy vim phi v lm cht chut bt ngun tth nghim bin np ca F. Grifith nm 1928) vo mi trng nui cy v kt hp vi vic x l bng DNase. iu chng tDNA quyt nh tnh cht di truyn ca phcu khun. Nm 1952, Alfred Day Hershey (1908 - 1997) v Martha Chase (1927 - 2003) cho bit, bng ng v phng x c ththeo di s di chuyn ca protein v DNA ca virus (csl DNA cha phosphor, protein cha lu hunh m khng cha phosphor; v vy DNA virus c th c nh du bng ng vphng xphosphor, cn protein virus c nh du bng ng v phng x lu hunh). Khi nh du mt sht virus phn protein v mt s ht virus phn DNA ri a vo mi trng nui cy vi khun ch E.coli th thy DNA virus nhanh chng thm nhp vo tbo ch, cn phn protein th khng. Th nghim cng chng minh tm quan trng vmt di truyn ca DNA. Nm 1955, nh ha sinh hc MFraenkel-Konrad (1910 - ) chia virus ra lm hai phn v sau ni c hai phn li, phn protein khng gy nhim (khng chui vo tbo vt ch) cn phn DNA th gy nhim. Th nghim ca Krauss trn hng cu ngi chng minh slin quan gia cu trc ha hc ca protein vi vai tr di truyn ca DNA (khi a DNA chit xut thng cu non ca ngi lnh vo ty xng bnh nhn thiu mu hnh li lim (c HbS khng bnh thng) th thy hemoglobin bnh thng c tng hp. iu chng tDNA quyt nh cu trc c hiu ca protein. Cc th nghim sau ca cc nh khoa hc ch trng vo vic tng hp DNA. Nm 1956 Severo Ochoa (1905 - 1993) v Arthur Kornberg(1918 - ) - hai nh khoa hc ngi M tng hp DNA in vitro bng cch trn enzyme DNA polymerase I c chit xut t E. colivi cc dNTP, DNA khun (DNA tnhin) v Mg++, DNA thu c khng khc DNA tnhin. Hai nh khoa hc ny c nhn gii thng Nobel vy hc v sinh l hc vo nm 1959. Nm nm sau khi cng bcng trnh cu trc xon kp caDNA vo nm 1958, Matthew Stanley Meselson (1930 - ) v Franklin William Stahl (1928 - ) chng minh bng thc nghim d on trn l hon ton c cs. Bng cch nui vi khun E.coli mi trng cha nit nng 15N sau bng 14N bnh thng ri theo di qua cc thh, xem s thay i ttrng ca DNA qua my ly tm siu tc (siu ly tm); hai nh khoa hc xc nh c c ch ti sinh bn bo tn ca DNA.

Vvic nghin cu RNA, cho n cui nhng nm 30 ca th kXX, cc nh khoa hc vn nghrng DNA l c trng ca cc t bo ng vt, cn RNA ch c th gp c trong nm men v cc t bo thc vt. Ngun quan trng nht ca DNA lc ny l tuyn c (thymus), cn i vi RNA l nm men. Cc nghin cu chnh xc hn sau ny cho thy c hai loi nucleic acid u c mt trong tt ccc tbo ng vt v thcvt. Nm 1939, cc nh nghin cu Torbjorn Oskar Caspersson(Thy in; 1910 - 1997) v Jean Brachet (B; 1909 - 1998) chng minh stng hp mnh mprotein trong bo tng cng xy ra vi stng hp manh mRNA. Nm 1956, S. Ochoa (nh ha sinh hc ngi M) dng enzyme RNA polymerase c chit xut tvi khun Azobacter vineladni tng hp RNA in vitro. Nm 1956, George EmilePalade (1912 - ) chng minh microsome c cha nhng ht c nhiu RNA gi l thribo tc l ribosome (55% RNA, cn li l protein). Nm 1961, Francois Jacob (1920 - ) v Jacques Lucien Monod (1910 - 1976) nu vn c mt loi RNA khc c c im chuyn ha nhanh chng v c cu to tng t DNA. l mRNA, l cht trung gian chuyn thng tin tDNA n chui polypeptide c tng hp. Trc vo nm 1958, M. B. Hoagland (nh ha sinh hc ngi M) nghin cu vn a amino acid vo ribosome. ng xc nh l trc khi tham gia tng hp protein, amino acid c kt hp vi RNA vn chuyn (tRNA) vn chuyn amino acid tbo tng vo ribosome. Vi nhng phng php th nghim khc nhau cc phng th nghim ca Marshall Warren Nirenberg (1927 - ), Har Gobind Khorana (1922 - ) v Ochoa xc nh c cc bba mt m ca ton b 20 amino acid vo nhng nm 1961 - 1965.Do vai tr quan trng ca nucleic acid trong hot ng sng ca cthsinh vt (tham gia vo cc qu trnh c bn ca s sng nh sinh tng hp protein, sinh trng, sinh sn v di truyn), cho nn vic nghin cu nucleic acid ngy cng c ch trng. T nhng nm 70 ca th k XX tr li y, nht l trong 10 nm cui ca thkv nhng nm u ca thkXXI ny, hiu bit v nucleic acid ngy cng c mrng. Nh nhng thnh tu ca sinh hc phn t, mt lnh vc khoa hc mi hnh thnh v pht trin mnh m, l cng ngh gen, mt trng tm c u tnghin cu hng u ca cng ngh sinh hc. 2. Cc phng php nghin cu: Cng nh s nghin cu cc i phn t sinh hc khc, c nhiu phng php v kthut c sdng trong vic nghin cu nucleic acid. Tuy nucleic acid c cu trc phc tp v hot ng rt cht ch, nhng nhnhng thnh tu mi vkthut, ra i nhiu phng tin v phng php nghin cu hin i, m bo chnh xc cao, cho php khm ph thm nhiu iu quan trng nucleic acid. 1. Cc phng php chung 1.1. Phng php nhiu xRnghen Phng php ny da trn stn xca tia X (tia Rnghen) qua cu trc tinh thca cht cn phn tch. Bng cch v pha ca tia X bnhiu xkt hp vi vic xl dliu trn my tnh, ngi ta c thxc nh c chnh xc vtr ca mt nguyn t bt k so vi cc nguyn tcn li trong mt i phn t. Nh vy, ngi ta c ththu nhn c mt hnh nh "tnh" vcu trc phn tnm trng thi tinh th. Tuy nhin, cn ch

rng cc cht nm trong tbo khng bao gitn ti trng thi tinh th, m chng thng nm trng thi ho tan hoc kt hp vi cc cu trc khc ca tbo. Bi vy, ngi ta thng sdng thm phng php phn tch cng hng tht nhn thu c cu trc phn tca cht cn phn tch trng thi hot ng sinh hc. Nh kt qu nhiu x Rnghen trn cc si nucleic acid, ngi ta xc nh c cu trc khng gian ca phn t nucleic acid (chyu l ca DNA). 1.2. Phng php in di Nguyn tc ca phng php ny l da vo tnh phn cc ca phn tv khnng di chuyn vmt hng xc nh khi chu tc ng ca mt in trng. Trong cc dung dch kim v trung tnh, cc i phn tnucleic acid tch in m ng u trn khp b mt nn trong in trng, chng s di chuyn vcc dng ca in trng. Bng phng php in di, chng ta c ththu c nucleic acid dng tinh khit t dch chit nghin cu. Phng php ny cng c dng tch ring cc nucleic acid DNA, RNA, cng nhcc loi RNA ring bit (mRNA, rRNA, tRNA). 1.3. Phng php sc k Phng php sc k da vo kh nng phn b ca cc cht chy sc k khi a vo dung mi kt hp vi khnng hp ph ca vt liu sc k. Cc cht khc nhau c khnng ha tan vo dung mi khc nhau v c vt liu sc k hp phvi lc khc nhau cho nn tc di chuyn ca chng trn sc k l khc nhau. Do cc cht ny s c tch ra trong qu trnh chy sc k. Phng php ny c dng tch chit v tinh chnucleic acid. C nhiu phng php sc k: sc k giy, sc k ct, sc k trao i ion, sc k lp mng, sc k kh. 1.4. Phng php quang ph Nguyn tc ca phng php ny l da vo s hp th mnh nh sng ca mt cht mt bc sng xc nh. Nucleic acid hp th mnh nh sng tngoi bc sng 260 nm do s c mt ca base purine v pyrimidine. Gi trmt quang bc sng 260nm (OD260nm) ca cc mu cho php xc nh nng nucleic acid trong mu. Ngi ta cng xc nh c tinh khit ca ch phm nucleic acid khi xc nh hp thnh sng 280 v 260 nm (hp thcc i ca protein l 280 nm). 1.5. Phng php ng vphng x Nguyn tc ca phng php ny l cc cht ng v phng x c gn ln cht nghin cu v dng kthut phng x t ghi theo di qu trnh bin i ca cc cht khi a chng vo c th hoc mi trng cn nghin cu. Nhvy c thxc nh c c ch tham gia qu trnh bin i ca cht cn nghin cu. Cng c th dng my o phng x nh lng cht cn nghin cu. Phng php ng vphng x c dng nghin cu c ch tng hp nucleic acid. Ngoi ra vic xc nh thnh phn cu trc cng nh nh lng nucleic acid cng c tin hnh bng phng php ny. Cc ng vhay dng cho cc nghin cu ny l: 3H, 14C, 32P, 15N. 1.6. Phng php ha hc Dng phng php thy phn thch hp bng acid hay kim, ngi ta c ththu nhn c cc phn khc nhau ca nucleic acid. Bng cch kt hp vi cc phng php khc nh sc k hay in di, cc thnh phn c tch ring nghin cu cu trc, thnh phn ha hc v tnh cht ca chng. Ngi ta cng c thdng phng php ha hc nh tnh nucleic acid trong t bo nh nhng phn ng ha hc c trng. 1.7. Phng php dng hthng v bo

Ngi ta c th s dng i tng nghin cu l c th ton vn, lt ct m, tbo, nhng mt phng php nghin cu rt c hiu qu l phng php dng cc hthng v bo (khng dng t bo nguyn vn). Nghin v t bo ri dng cc phng php ly tm tch cc thnh phn khc nhau ca t bo. Da trn nhu cu nghin cu, ngi ta c ththm vo hthng v bo cc cht khc nhau. Nh h thng v bo ny chng ta c thnghin cu qu trnh sinh tng hp protein, vai tr ca cc nucleic acid trong qu trnh ny, cng nhbn thn qu trnh tng hp cc nucleic acid. P. C. Zamecnik v cng sln u tin pht trin cc hthng v bo nghin cu sinh tng hp protein, xc nh cc ht ribonucleoprotein (ribosome) l ni sinh tng hp protein v pht hin ra tRNA. 2. Cc phng php tch chit nucleic acid m bo cho cc nghin cu tip theo, nucleic acid cn c tch vi mt lng ln v tinh sch. Chnh v vy iu cn ch trc ht l phi thu nhn cc nucleic acid trng thi nguyn vn, khng b phn hy bi cc tc nhn c hc hay ha hc. Vic nghin lc mnh khng ng quy trnh cng d lm tn thng n phn t nucleic acid (d b gy). Cc enzyme ni bo b ph v cng c th thy phn nucleic acid. c ch hot ng ca cc enzyme ni bo (deoxyribonuclease 15 = DNase, v ribonuclease = RNase) cn tch chit nucleic acid nhit thp, hoc s dng cc cht c ch s hot ng ca cc enzyme ny. 2.1. Phng php tch chit DNA DNA l phn tc kch thc ln nn cn trnh cc tc nhn c hc hoc ha hc mnh, trnh lm t gy. C nhiu phng php khc nhau c sdng tch chit DNA, nh: ly tm gradient ttrng ClCs, ct sc k trao i anion (Kit QIAGEN), v.v. Di y chl mt trong cc phng php tch chit DNA . Phng php tch chit ny gm 3 bc cbn sau: - Bc 1: Ph vmng tbo v mng nhn ( t bo eukaryote) bng hn hp cht ty (SDS - sodium dodecyl sulfate, sarcosyl) v proteinase. Cc DNA s c gii phng ra mi trng. Cc protein lin kt vi DNA cng t b phn hy. - Bc 2: Loi bthnh phn khng mong mun trong mu m chyu l protein bng dung dch: phenol chloroform. Dung dch ny lm bin tnh protein v khng ha tan nucleic acid. Sau khi ly tm loi protein (nm gia pha nc v pha phenol: chloroform) s thu c nucleic acid (pha nc). - Bc 3: Kt ta nucleic acid. Mc ch l thu nhn nucleic acid di dng c c v bo vchng khi sphn hy ca cc enzyme cng nhc thha tan trli theo nng mong mun. + Dng etanol nng cao (2,5 dung tch ethanol/1 dung tch mu) trong mi trng c lc ion cao (nng mui cao), nhit thp. Hu nh ton b nucleic acid u kt ta trong iu kin ny. + Dng isopropanol (thtch dung mi: thtch mu l 1:1), khng c shin din ca mui. Cc DNA trng lng phn t thp khng bkt ta, do c thloi chng ra khi dch chit bng cch ny. Ta thu c bng 2 cch trn c thu nhn li bng cch ly tm. Sau c ra li bng ethanol 70% loi bcc mui v cc du vt isopropanol. V cui cng l, tin hnh x l bng RNase loi bRNA. 2.2. Phng php tch chit RNA ton phn v mRNA Cc loi RNA u l cc phn t khng bn, d b phn hy bi cc enzyme ribonuclease (RNase). Hot tnh ca cc enzyme ny rt cao v bn vng vi cc tc

nhn thng dng bt hot enzyme (nhxl 900C trong 1h khng lm mt hot tnh n). RNase li c mt khp ni (v dtrn u ngn tay ca ngi thao tc...). Chnh v vy, cn phi c nhiu bin php thn trng trnh cc tp nhim bi cc RNase t mi trng: thao tc trong iu kin v trng, mi dng c ha cht u c khtrng bng nhit hay c xl vi DEPC hu trnh mi tip xc vi dng c bng tay trn. - Vic tch chit RNA ton phn cng gm 3 bc c bn nh i vi DNA: (Lu rng, y cng chl mt trong cc phng php c sdng m thi. Hin nay phng php c sdng phbin nht chit tch RNA l Tri201.) + Nghin tbo m trong dung dch c mt mt cht ty mnh (SDS, sarcosyl) nng cao, mt tc nhn gy bin tnh protein mnh (guamidium thiocyanate), mt cht kh(2 - mercaptoethanol). Cc cht ny c tc dng c chhot ng ca cc RNase ni bo v tch cc protein lin kt khi phn tRNA. + Loi protein bng xl phenol: chloroform v ly tm. + RNA c kt ta bng ethanol v thu li bng ly tm. Di dng kt ta trong etanol hoc ng lnh -700C trong nc c cha cht c ch RNase l R - nasine, RNA c th c bo qun trn mt nm. V, cui cng l bc tch RNA khi DNA. - Tch chit mRNA (cc tbo nhn chun) Khong 90-99% tng sRNA tbo l rRNA (80 - 85%), tRNA(15 - 20%), snRNA (<1%). Chng c kch thc v trnh txc nh v c th c tch ring bng in di, ly tm.... mRNA chim khong 1 - 5% tng s RNA tbo. Kch thc v trnh tca loi ny v cng a dng. Tuy vy, chng c mt c im chung l c cu trc ui polyA (c thln n 100A). Ngi ta c th tch mRNA ra khi mu bng cch da vo cu trc trn v c tnh lin kt bsung A-T ca nucleic acid, sdng sc k i lc trn ct oligodT - cellulose. Cc bmu th(cc kit) c xut hin trn thng trng hin nay sdng cc vin bi t c mang oligodT trn bmt l da vo nguyn tc nu trn. Rng lin kt b sung vi oligodT, sau khi cc mRNA bm ln b mt cc vin bi t, chng s c thu nhn li qua ly tm hoc s dng nam chm v mRNA s c tch khi cc vin bi v gili. Bng cch ny c ththu nhn mRNA tnhng mu c khi lng rt nh. Sau khi tch chit cc nucleic acid c th c tinh sch bng cc phng php siu ly tm, sc k hay in di. 2.3. Phng php siu ly tm Siu ly tm trn mt gradient lin tc cesium chloride (CsCl). Khi ly tm, dung dch CsCl m c trong ng ly tm st ng hnh thnh mt graient ttrng vi ttrng tng dn t ming ng xung y ng. Di tc dng ca lc ly tm, cc nucleic acid di chuyn trong ng n vtr c ttrng bng vi t trng ca chnh chng s t thcn bng v ngng li, hnh thnh mt lp c nh trong ng. Sau khi ly tm, lp ny s c thu nhn li. Phng php ny thng c dng tinh sch plasmid v phage. - Siu ly tm trn m CsCl (gradient khng lin tc): Hn hp nhiu nucleic acid c t trng bit trc khc nhau c t trn mt lp dung dch CsCl (m). Trong qu trnh ly tm ch c nhng phn t c t trng cao hn t trng lp m mi di chuyn qua c lp m. y, ng ly tm bao gm nhiu lp m c ttrng tng dn tming n y ng. Nucleic acid cn tinh sch s nm mt phn cch hai lp m. Phng php ny thng dng tinh sch mt lng ln phage. - Siu ly tm trn gradient saccharose: Thng dng phn tch th mt hn hp c kch thc chnh lch nhau nhiu kb. ng dng chng trong chn lc cc on DNA c kch thc xc nh dng trong vic thit lp cc ngn hng gen. 2.4. Phng php sc k

C thdng cc phng php sc k khc nhau phc v cho cc mc ch khc nhau trong tch chit nucleic acid. - Sc k i lc trn polyU - sepharose hay trn oligodT - cellulose tinh sch mRNA. - Sc k lc gel trong phn tch cc nucleic acid ra khi cc nucleic tdo sau qu trnh nh du DNA hoc RNA. - Sc k trao i ion trn vi ct thu hi nhng lng rt nhDNA. - Sc k lng cao p (High Performance Liquid Chromatography - HPLC). Phng php c phn gii cao ny c dng trong tinh sch cc oligonucleotide tng hp (phn gii mt nucleotide), plasmid, phn tch cc on DNA. 3. Cc phng php phn tch nh tnh v nh lng th nucleic acid Mun nh tnh v nh lng cc nucleic acid, ngi ta phi tin hnh thu nhn chng dng sch. Cc phng php thng dng trong nh tnh v nh lng chng l phng php o mt quang, in di, siu ly tm, sc k. 3.1. Phng php nh lng bng quang phk Phng php ny cho php c lng tng i nng nucleic acid c trong mu phc vyu cu nghin cu. Nguyn tc ca phng php c trnh by phn trc. kim tra sch ca dung dch, ngi ta o thm gi trOD 280 nm (OD280nm). bc sng ny, cc protein c mc hp thcao nht. Ngoi ra, cc protein cng hp th nh sng bc sng 260 nm nhcc nucleic acid v do lm sai lch gi tr tht ca nng nucleic acid. Mt dung dch nucleic acid c xem l sch (khng tp nhim protein) khi tsOD260nm/OD280nm i vi DNA l OD260nm/OD280nm 1,8; cn i vi RNA l OD260nm/OD280nm2. 3.2. Phng php in di gel Csca phng php ny c trnh by phn trc. Hai loi gel c sdng trong nghin cu nucleic acid l gel polyacrylamide v gel agarose. Vic chn loi gel cng nh nng cc cht to thnh gel ty thuc kch thc trung bnh ca cc on nucleic acid cn phn tch. Mi tng quan gia nng agarose v acrylamide cn sdng phn tch cc trnh tc kch thc xc nh c thhin qua bng 1.1. 3.2.1. Gel polyacrylamide Thng dng tch cc on kch thc nhdi 1000 cp base. So vi gel agarose th thao tc vi gel ny phc tp hn. V vy, gel ny ch c dng cho nhng mc ch c hiu. ng dng chyu ca loi gel ny l: - Tinh sch cc oligonucleotide tng hp - Xc nh trnh t DNA - Tch cc on DNA c chiu di di 500 cp base Gel c gia hai tm thy tinh v in di thc hin theo chiu thng ng. 3.2.2. Gel agarose L loi gel thng dng nht. Thao tc vi loi gel ny n gin, thng dng phn tch nhng on c kch thc trong khong 0,5 - 20kb. Gel c trn mt gi thnm ngang v in di c thc hin theo phng nm ngang. Trong gel agarose cc nucleic acid shin hnh di tia tngoi (UV) nhethidium bromide. Ha cht ny c kh nng gn xen vo gia cc base ca nucleic acid v spht hunh quang di tc dng ca tia t ngoi. Di schiu sng bng tia t ngoi (= 260 - 360 nm), nucleic acid shin hnh di dng nhng vch mu cam.

 c lng kch thc cc trnh tnucleic acid trong gel agarose ngi ta sdng mt yu t nh du trng lng phn t(molecular weight marker - MWM). l tp hp nhiu trnh tDNA c kch thc bit (thang DNA - DNA ladder). in di trn gel agarose cn c sdng tinh sch v thu nhn mu. y, sau khi in di, cc vch tng ng vi DNA cn tinh sch c pht hin v thu nhn li theo mt trong cc phng php sau: (i) Ct phn agarose cha cc vch DNA, sau thu nhn li DNA bng cch khuych tn tgel agarose vo mt dung dch m thch hp. (ii) Khot mt ging nhtrong agarose ngay di vch DNA. Bm dung dch m vo y ging v in trng c ti lp. DNA di chuyn vo ging cha y dung dch m v c thu nhn li. (iii) Dng gel agarose c bit nhNusieve hay Seaplaque c im nng chy rt thp (650C) cho in di. Sau in di, vch DNA c ct ra v trong mt dung dch m nhit 650C. Khi agarose hon ton tan chy DNA c thu nhn li sau nhiu cng on tch chit v kt ta. 4. Cc phng php xc nh trnh tca nucleic acid Cc phng php phn tch nucleic acid nu cung cp nhiu thng tin v nucleic acid nhng cha cho php kt lun vbn cht ca mt on nucleic acid cth. Thng tin v s tng ng ca nucleic acid vi gene g, c chc nng iu ha hay m ha cho protein no, chc thrt ra c tvic xc nh trnh tnucleotide ca nucleic acid. Cc phng php xc nh trnh tnucleic acid ni chung da vo hai nguyn tc cbn, c tm tt di y: - Nguyn tc ha hc (Phng php Maxam - Gilbert, 1977): da vo cc phn ng ha hc thy phn c hiu phn tDNA, to thnh mt tp hp nhiu phn on c kch thc khc nhau. - Nguyn tc enzyme hc (Phng php Sanger (1977) v cc phng php ci bin): Da vo stng hp mch bsung cho trnh tcn xc nh nhDNA polymerase. Vi vic sdng thm dideoxynucleotide (nhm 3-ON c thay bng H) cng vi cc deoxy nucleiotide thng thng, kt qutng hp cng l shnh thnh mt tp hp nhiu on DNA c kch thc khc nhau. c hai trng hp, cc phn on DNA s c phn tch qua in di trn gel polyacrylamide c khnng phn tch hai trnh t DNA chchnh nhau mt nucleotide. Vi vic s dng mt on nucleotide c nh du ng vphng x, kt qu trnh t cn xc nh dc c trn bn phng x t ghi t bn in di.

III. Lipit:
1. Lch s nghin cu: Overton (1899) thy rng, nu mt cht ha tan trong dung mi khng phn cc th cht y d thm vo bn trong t bo. Do , mng phi c cu to bi cc phn t k nc. Theo Gorter v Grendel (1925), v cc lipit to mt lp n phn t trn mt trung gian nc - kh, nn chng phi c t chc thnh lp i trong mng. Khi , phn c cc ca mi lp n hng v mi trng nc (mi trng ngoi t bo v mi trng t bo cht), phn v cc xa hai lp mng n i din nhau gia mng.

Theo hai m hnh trn, lipit ca mng lin h trc tip vi mi trng ngoi. Tuy nhin, sc cng b mt ca mng rt thp sp vi trung gian lipit/nc. Do , Danielli v Davson (1935) co rng c mt lp n protein hnh cu ngm nc trn mi lp lipit. Cc protein ny tng tc vi mi trng nc xung quanh v phn hu cc ca lipit (do lm gim sc cng b mt. Robertson (1955) ngh m hnh lp i lipit vi cc protein hnh si t ln trn. Hn na, do tnh thm mnh ca vi phn t thch nc c kch thc nh (nc, methanol), nn ng ngh ti cc ng a nc to bi cc protein hnh si xuyn mng. 2. Phng php phn tch nh lng: Xc nh thnh phn axit bo c trong du m bng pp sc k kh: Vic phn tch thnh phn axit bo ca mt cht bo cho chng ta bit bn cht ca cht bo l loi du g hoc m g bi v mi loi cht bo khc nhau c c trng bi thnh phn axit bo khc nhau. Thnh phn axit bo ca du thc vt hoc m ng vt thay i ty theo ging, a im trng trt hoc chn nui, tui hoc ma thu hoch. Xc nh c s bin i hm lng v thnh phn axit bo c trong du m; t xc nh c ch s axit c trng cho sn phm du m. Vi cc loi du m thc phm ch s axit khng c vt qu 2.25, nu vt qua mc an ton ny, cn phi tinh ch h thp ch s axit bng bin php trung ha bi kim v khi ch s ny c dng lm c s tnh ton lng kim cn thit trung ha. Xc nh hm lng cht khng x phng ha: Nhng cht khng x phng ha trong du thc vt l nhng cht khng tc dng vi kim, thng l cc sterol, cc ru c phn t ln, cacbuahydro v cc cht mu. N biu th phm cht v tinh khit ca du. Dng kim x phng ha du. Sau dng ete petrol chit cc cht khng x phng ha ra khi dung dch x phng. Phng php nh lng cholesterol: Nguyn tc ca phng php l phn ng ca cholesterol v cholesterol este vi anhydrit axetic v nng axt sulfuric m c, dn n s hnh thnh ca mt phc hp mu xanh-mu xanh l cy. V vy, c th xc nh lng cholesterol trong chit xut lipid vi mt ng cong tiu chun ca nng cholesterol c bit n. PHNG PHP FOLCH Phng php ny dng xc nh hm lng cht bo ca cc sn phm rn v lng.