Chng 6 K THUT SINH HC PHN T TRONG PHN TCH H GENE SINH VT

Tm tt: Sinh hc phn t l nng ct ca cng ngh sinh hc. Phn tch sinh hc phn t c bt u t vic thu nhn dch chit ADN t t bo sng sch thc hin cc phn tch tip theo. Tu theo vt liu nghin cu m c phng php tch chit ADN ph hp. Dung dch ADN sau khi kim tra hm lng v sch c s dng phn tch theo nhng mc ch khc nhau nh Southem blot, Northem blot, xc nh trnh t ADN, PCR, RFLP, AFLP,... Ni dung ca chng gm 5 vn c bn: (1). Phng php tch chit ADN, (2). Lai phn t, (3). Xc ih trnh t nucleotit, (4). K thut RFLP, (5). Trnh t nucteotit lp li. Thnh tu ca sinh hc hin i v nhng hiu bit v di truyn hc phn t lm c s cho vic nghin cu, xy dng cc k thut sinh hc hin i. Nhng k thut sinh hc phn t ng dng trong phn tch genome ca sinh vt c cng b trong cc tp ch khoa hc v cc sch chuyn kho.

1. PHNG PHP TCH CHIT ADN Mi nghin cu v ng dng sinh hc phn t u bt u bng vic thu nhn mt lng axit nucleic, tinh sch tin hnh cc th nghim tip theo. iu quan tm hng u l cc k thut tch chit axit nucleic l thu nhn cc phn t trng thi nguyn vn khng b phn hu bi cc tc nhn c hc hoc ho hc. C th ly v d cc bc tch ADN t mm ht u di dy:

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Hnh 6.1. S quy trnh tch chit ADN t mm ht u tng v u xanh T v d c th trn ta thy quy trnh tch chit ADN gm ba bc c bn sau: Bc 1: Ph mng t bo v mng nhn (t bo Eukaryot). Thng thng ngi ta nghin t bo, m trong hn hp cht ty (SDS, sarcosyl) v proteinase K. Hn hp ny ph v mng t bo v mng nhn, gii phng ADN ra mi trng ng thi phn hu cc protein. Bc 2: Loi b cc thnh phn khng mong mun, ch yu l protein bng li tm. Bc 3: Ta axit nucleic trong ethanol hoc trong isopropanol thu nhn c axit nucleic di dng c c. Sau khi thu nhn axit nucleic dng sch, ngi ta tin hnh phn tch nh tnh v nh lng chng bng mt s phng php nh o mt quang, in i, siu li tm, sc k (hnh 6.2 v 6.3).

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Hnh 6.2. Ph hp th ADN bc sng 260 nm in di ADN l k thut c s dng kim tra cht lng ca ch phm ADN tch t t bo sng. ng thi kt qu in di cn cho php d on c hm lng ADN trong dung dch tch chit. Phng php in di dc s dng kim tra sn phm PCR, gii trnh t ADN...

Hnh 6.3. Cu trc ca agarose (theo Nng Vn Hi, 2002) Gel agarose c s dng phn tch in di ADN. Agarose dng bt dc pha vi dung dch TBE hoc TAE v tu theo mc ch phn tch ADN m pha gel vi nng thch hp. K thut in di ADN bao gm cc bc (hnh 6.4):

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Hnh 6.4. Cc bc to gel in di agarose (1, 2) To khun ga agarose; (3) ga trn khun ga ; (4) Tra dung dch ADN ; (5) Chy in di. Sn phm in di ADN c nhum bi EtBr v chp nh di nh sng n cc tm. (1) Nhum gel bng EtBr ; (2) Soi gel trn nh sng n cc tm pht hin s c mt ca ADN ; (3) Chp nh phn tch.

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Hnh 6.5. Hnh nh in di ADN tch t mm u tng 1. ADN chun 2, 3, 4, 5, 6, 7, 8: ADN tch t mm u tng

Hnh 6.6. Cu trc ca EtBr (theo Nng Vn Hi, 2002)

2. LAI PHN T Mc ch ca k thut ny l pht hin trnh t nucleotit c th, nghin cu cu trc ADN h gene (gencomic ADN). Phn t ADN b phn ct bi enzyme gii hn thnh nhng phn on ADN ring bit. Bng phng php in di cc phn on ADN ny vi nhng kch thc khc nhau c tch ra (separate) trn gel thch hoc hoc poliacrylamit v c chuyn sang mng lai phn t. Qu trnh lai phn t xy ra gia phn t ADN v on d ADN c gn nhn phng x theo quy tc lin kt b tr. Cc on ADN c th c pht hin theo phng php thm tch ca Southern v phng php lai phn t vi on d ADN c trnh t nucleotit tng ng. Phng php t ghi phng x (autoradiogrophy) c th nhn bit v tr on d ADN trn mng lai phn t. T khi E.M Southern thuc Trng i hc tng hp Edinburgh pht hin ra k thut ny (1975) gi l k thut phn tch mng thm truyn Southern (Southern blot analisis) gi tt l Southern, n c ng dng rng ri trong cc lnh vc nghin cu sinh hc phn t v di truyn. Khi ng dng k thut Southern i vi ARN th gi l Northern, cn vi protein th c gi l Western. Hin nay c nhiu ci tin v phng php, hnh thnh nhiu k thut phi hp nh Southwestern.
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2.1. Phng php Southern blot S dng mt trnh t ADN bit trc lai vi on ADN cn tm. Cc bc tin hnh c th thc hin theo cc bc sau: - Ct ADN bi enzyme gii hn thnh cc on ADN nh c th dch chuyn trn gel agasore. - Bin tnh cc si ADN kp trn gel agarose bng phng php x l vi kim. - Chuyn ADN bin tnh sang mng nylon hoc nitrocellulose. - Lai vi trnh t ADN c nh du bng phng x hoc hunh quang.

Hnh 6.7. S m t phng php Southern blot Phng php Southern blot cho php xc nh c trnh t on ADN cn tm, c th pht hin c t bin mt on hoc thm on khi so snh c th t bin vi c th bnh thng. 2.2. Phng php Northern blot Phng php Northern blot tng t nh phng php Southern blt, nhng mc tiu ca phng php Northern blot l nghin cu cu trc ca mARN. l k thut lai gia mARN vi trnh t ADN bit trc, nh du phng x. (1) Tch chit ARN tng s v bin tnh. (2). ARN c chuyn sang mng v lai vi trnh t ADN c hiu. (3). V tr v s lng cc on ca ARN lai vi ADN c pht hin trn phim phng x. Phng php Northern blot cho php nghin cu cu trc ARN, pht hin t bin.

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3. XC NH TRNH T NUCLEOTIT CA ADN C nhiu k thut xc nh trnh t cc i baz trong mt chui ADN: - K thut in i phn on nh du u cui cho php xc nh trnh t chui ADN si n. - Xc nh trnh t nucleotit thng qua trnh t amino axit da vo cc b ba m ho, tuy nhin s m ca cc amino axit thng c mt s nucleotit trng lp hoc mt s b ba cng quy nh mt loi amino axit, cho nn trnh t ADN thu c ch l tng i. - Xc nh trc tip thng qua phn ng kt thc u cui. Nguyn tc chnh l to ADN si n, m phn t sau c cng trnh t v ch di hn phn t trc mt nucleotit mt u cui. Gene t bo tin nhn c trnh t lin tc, cn gene sinh vt nhn thc c th gin on. on nucleotit m ho protein c gi l exon, cn on khng m ho gi l intron. 3.1. Phng php ho hc ca Maxam-Gilbert Nguyn tc ca phng php ho hc ca Maxam-Gilbert l s dng phng php nh du phng x (P32), x l ho hc ph hu 1 nucleotit to ra hng lot cc on ADN c kch thc khc nhau.

Cc on DNA b ph hu 1 nucleotit in di trn gel poliacrylamid Hnh 6.8. S minh ho phng php Maxam-Gilbert Cc khu trong phng php Maxam-Gilbert : (1). ADN si kp bin tnh thnh dng si n. (2). nh du phng x (P32) u 5'. (3). S dng 4 loi ho cht khc nhau, mi loi ho cht ch ph hu mt loi nucleotit nht nh v tin hnh 4 phn ng c lp, kt qa to ra cc on ADN kch thc khc nhau.
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(4). Sn phm ca 4 phn ng c tin hnh in di trn gel poliacrylamid. Trnh t nucleotit c c t di ln theo chiu t 5'3'. 3.2. Phng php Sanger Nguyn tc ca phng php ny l s dng deoxynucleotit khng c nhm OH v tr 3' do vy trong qu trnh ko di chui polinucleotit enzyme polimerase gn chng vo chui polinucleotit th qu trnh tng hp b dng li. Kt qu to ra cc on ADN c kch thc hn km nhau mt nucleotit, trn c s xc nh c trnh t nucleotit.

Hnh 6.9. Deoxynucleotit mt oxy v tr C 3' ca phn t ng Cc khu ca phng php Sanger: (1) Bin tnh ADN si kp thnh ADN 2 si n. (2). Mi tip hp vi ADN si n. (3). Phn ng tng hp chui polinucleotit gm: ADN si khun, primer, ADN polimerase v dNTP. S gn dNTP mt nhm OH v tr 3' vo chui polinucleotit lm qu trnh tng hp b dng li. (4). in di trn gel poliacrylamid s xc nh c trnh t ca on ADN.

Hnh 6.10. S xc nh trnh t nucleotit theo Sanger

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4. RFLP TECHNOLOGY (K thut phn tch hin tng a hnh ca di cc phn on ADN) Trong nhng nm gn y k thut phn lp gene dn thc y c bn trong lnh vc th v nht l vic s dng cc on ADN phn lp nh cc ch th phn t. K thut phn tch hin a hnh ca di cc phn on ADN l mt k thut mi m y ha hn lm thay i hon ton mt s lnh vc trong i truyn v chn ging. Thng tin di truyn c cha trong cc chui ADN ca cc nhim sc th nhn v b gene ca cc c quan t. Cy trng c kh nng sao chp phn t ADN vi ch chnh xc cao v mau l song c nhiu tc ng t nhin v nhn to gy ra bin d trong trnh t ADN. Nhng bin i v cp baz nit c th xut hin hoc do bin i chui ADN trn phm vi rng ln nh l kt qu ca s o on, chuyn on v khuyton hoc do chuyn ch trong nhim sc th. C mt s cch xc nh bin d ca chui ADN, l xc nh trnh t ADN v so snh tng on; s dng enzyme ct hn ch ct ADN thnh nhng phn on c chiu di khc nhau.

Hnh 6.11. Thit b t ng xc nh trinh t ADN mao qun Bin i trnh t nucleotit ti mt locus dn n xut hin bng ADN mi trn gel thch hoc poliacrylamid. Hin tng ny c gi l a hnh ca di cc phn on ADN. Hin tng a hnh di phn on ADN l cc ch th phn t. Bng cch s dng cc ch th phn t (ch th phn t RFLP) cc nh sinh hc phn t v di truyn chn ging c th thit lp cc bn gene lin kt di truyn v nh v cc gene c tm quan trng v kinh t. K thut RFLP c tin hnh theo cc bc sau:
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(1). Tch ADN t m sng. (2). S dng cc enzyme ct hn ch ct ADN thnh cc on c kch thc khc nhau. (3). in di trn gel agarose. (4). So snh cc on ADN gia cc i tng nghin cu. 4.1. Bn RFLP 4.1.1. Nguyn l Nguyn l ca k thut ny da trn s khc bit t nhin ca cc chui nucleotit trong phn t ADN v khi phn t ADN b ct thnh nhiu on nh bi enzyme ct hn ch th cc on ADN c th khc nhau v kch thc hay chiu di. S khc nhau ny c th c khai thc phn tch s phn li ca cc on nhim sc th khi lai to. Cc phn t ADN c kch thc nh nh ADN lc lp c th s dng enzyme (RE) gii hn lp bn RFLP, tuy nhin kh nng ng dng ADN lc lp trong to ging rt hn ch, v hu ht cc gene c ngha kinh t quan trng li nm trong nhn. Phn tch RFLP c th ng dng vi ADN nhn, nhng rt phc tp v ADN nhn c kch thc ln v hm lng nhiu. Khi s dng RE i vi ADN th hng triu phn on ADN c to thnh. 4.1.2. Th vin ch th RFLP Ngi ta dng nhng on nhim sc th lm ch th nh du nhn bit cc phn on ct hn ch. p dng phng php lai ADN - ADN c tnh c hiu cao bi vic s dng cc on ADN mu nhn bit cc phn on ADN nhn t hn hp cc on ADN to thnh bi vic x l RE. Tp hp cc on ADN mu c gi l th vin RFLP. on ADN mu c di 2-5 kb trng thi tinh khit v c s dng lai phn t. Cc on ADN tinh khit ngi ta cn phi thc hin k thut nhn gene (gene cloning). K thut nhn on ADN c th c hin nh sau: on ADN c gn vo plasmit ri bin np vo vi khun. Vi khun sinh sn dn n on ADN c nhn ln to nhiu bn sao on ADN. Bng k thut nui cy v phn lp plasmit t vi khun s to ra mt s lng ln cc on ADN dng lm mu cho lai ADN. Quy trnh xc nh RFLP c thc hin theo cc bc sau : (1) Tch chit ADN nhn ; (2). X l ADN vi enzyme ct gii hn ; (3). in di sn phn ct bi RE ; (4). Bin tnh ADN thnh dng si n; (5). Chuyn ln mng lai nitrocellulose (Southern blot) on nADN mu lai vi on ADN tng ng theo nguyn tc b sung gia cc cp baz. Ngi ta s dng k thut pht quang ho hc (Enhanced Chemical Luminesence = ECL) hoc nh du phng x xc nh kt qu lai bng phim m bn hoc bng phng x t ghi.
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4.1.3. Thit lp bn di truyn RFLP Ngi ta tin hnh lai hu tnh c F1 v cho F1 t th cho F2. Khi F1 gim phn hnh thnh giao t th cc nhim sc th c s ti t hp thng qua trao i cho. Trao i cho dn n ti t hp l c s cho vic thit lp bn di truyn truyn thng (bn lin kt). K thut RFLP cho php quan st trc tip cc phn t ch th ADN trn on nhim sc th. Cc ch th RFLP phn li hon ton chnh xc nh cc ch th gene v tun theo cc nh lut Menen mt cch nghim ngt. V th bn RFLP c thit lp theo nguyn tc nh i vi cc ch th truyn thng. Quy trnh thit lp bn di truyn bng ch th RFLP c thc hin theo cc bc sau: (1) Chn cp b m cng loi xa nhau v mt di truyn to F1. Tch ADN v s dng RE x l ADN. Sng lc tnh a dng v cp b m no c mc a dng thch hp c chn lm cp lai. (2). To qun th lp bn . Cho cp b m lai vi nhau c F1, cho F1 t phi c F2 hoc c th lai ngc. Mt qun th F2 hoc lai ngc c 50 c th th c th xy dng bn di truyn chi tit. (3). nh gi RFLP trong qun th. Tch ADN tng c th v tin hnh k thut RFLP. Qu trnh lp bn c tin hnh theo 3 qu trnh sau: - Sng lc RFLP: Mu c th vi cp b m pht hin ra loi enzyme gii hn thch hp to ra s a dng RFLP r rt nht gia hai c th b m. - Chn cp ch th 1enzyme: Chn cp ch th /enzyme thch hp, l loi enzyme ct ADN. C th tin hnh nh sau: tch ADN cy b, cy m v tng cy con lp bn . S dng tng loi enzyme ct, in di, thm truyn ln mng lai (mi enzyme dng mt b mng lai ring), chn cp ch th / enzyme th hin s a dng v kiu gene s dng lp bn . - nh gi mc lin kt: Khi so snh hai ch th thu c hai kt qu, (1). Chng c xu hng cng phn li v hai ch th c mi lin kt. V d: F2 d hp v ch th 1 v d hp v ch th 2 trong khi b m ng hp v c hai ch th. (2). S phn b tnh ng hp hay d hp ca hai ch th l khng theo quy lut m hon ton ngu nhin, nh vy chng khng lin kt. S dng nhiu ch th s xc nh c mc lin kt khc nhau ca tng ch th v nhm ch th. 4.2. ng dng ca RFLP Vic pht trin k thut phn tnh hin tng a hnh ca di cc phn on ADN m rng cnh ca i ti pht hin, theo di v iu khin s bin d di truyn nhiu loi cy trng m trc y khng th lm c (Tanksley
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v cng s, 1989). Nhng nghin cu gn y v la (Mc Couch v cng s, 1988), c chua (Pettersun v cng s, 1988, 1990, 1991), u tng (Keim v cng s, 1990), la m (Sharp v cng s, 1989), Ng (Burr v cng s, 1988, Grant v cng s 1989, Beavis v cng s. 1991, Stuber v cng s, 1992) chng t rng vic lp bn cc ch th phn t RFLP l mt phng php c sc mnh ln lao trong vic xc nh cc locus iu khin cc tnh trng s lng - cc c tnh c tm quan trng v mt nng nghip. Ngi ta cng xc nh nhiu tnh trng n gene cy la thng qua lin kt vi cc ch th phn t RFLP (Mackill v cng s, 1993, Ronald v cng s, 1992, Mc Couch v Tanksley, 1991, Yu v cng s, 1991, Yoshimura v cng s, 1992). K thut phn tch hin tng a hnh ca di cc phn on ADN cho php pht hin hin tng a hnh ca b gene thng qua so snh cc bn phn ct ADN gia cc c th khc nhau. Cc ch th phn t RFLP mang tnh cht ring bit, ng tri v khng gy hi. Cc ch th phn t ny khng chu tc ng bi iu kin mi trng (k c s thay i v ma v v v tr a l v giai on pht trin ca cy. Vic phn tch hin tng a hnh ca di cc phn on ADN c tin hnh trn phn t ADN tch chit t l non, trnh vic phi chm sc cy cn non ti lc trng thnh. Ngi ta p dng cc ch th phn t RFLP vo cng tc chn ging gin tip, lm rt ngn qu trnh chn lc cc gene qu him, cc gene c tm quan trng v kinh t. Bi l cc ch th phn t RFLP c th lin kt cht ch vi cc gene trn v vn c duy tr trong qu trnh phn li. V vy ngi ta c th theo di v chn lc cc gene thng qua cc ch th phn t . N. R. Apuyru v cng s (1988) xc nh hin tng a hnh ca di cc phn on ADN ca hai ging u tng hoang di Minsoy v Norin 1 bng cch s dng cc dng ADN chn lc ngu nhin lm on d ADN. Cc tc gi nhn thy rng mt trong s nm on d th nghim bc l tnh a hnh. Hn na s bng a hnh xut hin l do c s sp xp li phn t ADN. P. Knh v cng s (1989) kho st 58 ging u tng chn lc t Geneus Glicine v Subgeneus Soja bng 17 ch th phn t RFLP nh gi mc a dng phn t, tnh hu hiu ca cc ch th phn t c th hin t nht trong s cc ging u tng dng trng v n c biu hin nhiu nht gia cc ging thuc cc loi u tng khc nhau nh Glicine max (L) Merr, G. soja Sieb v Zucc. Cc ch th phn t RFLP c p dng cho vic lp bn gene, nghin cu a dng sinh hc, phn loi pht sinh chng loi cho n iu khin vt nui cy trng. N l mt cng c c lc trong cng tc lai to v chn ging thc
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vt. ng thi vic xc lp cc ch th phn t RFLP m ra mt hng mi l phn lp cc gene m vn cha bit sn phm ca n. THO LUN 1. Th no l lai phn t? Phn bit phng php Southern blot, Northern blot. Nhng ng dng ca cc phng php ny. 2. Vn xc nh trnh t ADN. Nguyn tc v phng php. 3. Vn xc nh ch th RFLP v bn RFLP.

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