Chng 2 CC K THUT NN CA CNG NGH SINH HC HIN I K thut di truyn hay cng ngh gen hay cng ngh

ADN ti t hp l cng ngh ct li ca CNSH hin i. N bao gm cc k thut thc hin trn phn t acid nucelic, nhm nghin cu cu trc ca gen; iu chnh, bin i gen; tch, tng hp v chuyn np gen vo t bo sinh vt ch mi to ra c th mi, sinh vt mi mang c tnh mi. K thut ny ra i da trn hng lot thnh t ca sinh hc phn t trong vic ct, tch, ni, ghp, chuyn v cho cc gen biu hin ng nh mong mun. 2.1. Cc khi nim chnh s dng trong cng ngh ADN ti t hp 2.1.1. ADN ti t hp l g? ADN ti t hp l ADN c cu thnh t hai hay nhiu on ADN c ngun gc khc nhau. 2.1.2. Cc enzyme dng trong cng ngh AND ti t hp 2.1.2.1. Cc enzyme gii hn (Restriction enzyme - RE) Nm 1962, V.Arber ln u tin chng minh rng c nhng enzym c bit hot ng trong t bo vi khun, chng c kh nng phn bit ADN ca mnh vi AND l ca phage, cc enzym ny hn ch s sinh sn ca phage trong t bo vi khun bng cch phn hu chng mt cch ch hiu, do c gi l restriction enzyme. Nm 1970, H.Smith v cs tch c RE u tin t vi khun Haemophilus influenzae (HinII), t n nay, hng lot RE c pht hin v tch chit thnh cng (BamHI, EcoRI, NcoI, XbaI,) Phn loi RE: (i). RE ct ti v tr cch v tr nhn bit khong 1000 1500 bp v pha cui ca phn t AND v gii phng khong vi chc cp bp. (ii). RE ct ngay ti v tr nhn bit ca mnh trn phn t AND. Loi ny c s dng ph bin hin nay. (iii) RE ct trc v tr nhn bit ca mnh khong 20 bp trn phn t AND. Cc RE loi ii: Cch gi tn: - ch u vit hoa l ch u tin trong tn chi loi vi khun m t cc RE c pht hin. - tip theo l 2 ch u ca tn loi khng vit hoa. - Mt ch vit hoa ch tn chng vi khun (nu c) - Ch s la m ch th t RE c pht hin. V d: RE HinII l enzyme th 2 c tch t vi khun Haemophilus influenzae; Cc kiu ct ca RE loi ii: Mi RE c trnh t nhn bit c trng (khong 4 8bps i xng o ngc nhau) trn phn t AND. Cc RE di chuyn theo chiu 5 3 trn 2 mch ca phn t AND v ct nhng im nhn bit c trng ca mnh. C 2 kiu ct ca cc RE: (i). Ct u bng: cc RE ct c 2 mch cng mt v tr 2 mch ca AND khng c kh nng t kt hp li vi nhau. (ii). Ct u so le (u dnh): Cc RE ct 2 mch ca AND 2 v tr lch nhau, kt qu to nn cc on AND c cc u so le vi trnh t nucleotit hon ton b sung cho nhau nn c th t ni li vi nhau.

V d: EcoRI ct: ----G A A T T C --------C T T A A G ----RE HaeIII ct: ----G A A T T C ------ C T T A A G ---

--------G G C C -------------C C G G ------

----------G G ----------C C

C C ---------G G ----------

Cc RE ct u dnh EcoRI, BamHI, NcoI, XbaI, TagI, khi ct cc AND mch kp s to ra cc u lch c trnh t nucleotit hon ton b sung vi nhau nn d dng bt cp gn li vi nhau nh lc cha b ct. V th, nu c mt on AND l khc cng b ct bi RE ny th on AND l ny c th xen vo gia phn t AND ban u v to nn AND ti t hp. y chnh l mt tnh cht rt quan trng ca cc RE. Vi cc RE ct phn t AND to ra u bng, cn phi c qu trnh gn thm mt vi nucleotit vo 2 u va b ct to ra u dnh, sau mi c th tin hnh gp ni vi AND l to ADN ti t hp . Ngoi ng dng to ADN ti t hp, cc RE cn c s dng trong vic xy dng bn ct gii hn ca phn t AND. iu ny c ngha v cng quan trng, t ngi ta c th xc nh c loi RE no cn c s dng trong cc nghin cu c th. Mt s loi RE Mt vn t ra l trong t bo vi khun lun tn ti cc RE, vy phi c mt c ch no bo v cc AND ca t bo vi khun cc AND ny khng b chnh cc RE ca mnh thu phn? Qua nghin cu, ngi ta thy rng trong t bo cn c mt loi enzyme sa i, enzyme ny c kh nng gn thm cc nhm methyl vo mt hay mt vi gc baz ca phn t AND (methyl ho phn t AND), lm cho RE xc nhn n khng phi l AND l nn khng phn gii n. 2.1.2.2. Cc ligase Chc nng: Ni 2 on ca phn t AND li vi nhau to nn ADN ti t hp. Thc cht, AND ligase xc tc s to thnh lin kt phosphodieste gia 2 on ca phn t AND. V nguyn tc, cc u dnh ca phn t AND khi c ct bi cc RE c th t bt cp tr li vi nhau, tuy nhin trong thc t hiu qu ca qu trnh t bt cp ny khng cao do lc kt dnh ca 4 bp t do ca cc u dnh khng mnh gi phn t AND ny ni li vi nhau. vic gn kt cc u ca phn t AND t hiu qu cao, cn phi s dng cc ligase, cc ligase s xc tc s hnh thnh lin kt ho tr gia u 3OH vi u 5(PO 4)-3 ca 2 u ca on phn t AND, to nn AND ti t hp. Ligase c s dng ph bin nht l AND ligase T4, c tch t vi khun E.coli b nhim thc khun th T4, ligase T4 c th gn cc on AND c u so le hoc u bng. 2.1.2.3. Cc polymerase Gm AND polymerase (xc tc cho qu trnh ti bn AND) v ARN polymerase (xc tc cho qu trnh tng hp ARN). Phn loi: AND polymerase c 3 loi: vi khun: - Loi I: loi b on ARN mi v tng hp on AND ngn v tr mi va b ct (phn on okazaki) - Loi II: xc nh s bt u v kt thc tng hp chui axit nucleic. -Loi III: gn cc nucleotit t do trong mi trng vo chui tng hp mi theo ng trnh t b sung v vi hot tnh exonuclease 5 3 v 3 5 nhm enzyme ny cn c chc nng kim sot s chnh xc trong qu trnh tng hp (ct b cc nucleotit gn sai v thay th vo chnh xc loi nu b sung vi mch khun). sinh vt nhn chun: - Loi : Ti bn AND ca nhn. - Loi : Sa i AND trong qu trnh ti bn. - Loi : Ch lm nhim v ti bn h gen ca ti th. Taq polymerase: l ADN polymerase chu nhit cao c tch chit t vi khun sui nc nng Thermophilus aquaticus. y l enzyme c s dng rt ph bin v hiu qu trong phn ng tng hp ADN trong iu kin invitro (PCR,

RADP,...). Taq polymerase c hot tnh ti u 720C, nhng n c th bn ti 940C, trong 1 giy taq polymerase gn c 75 nu vo chui polynucleotit ARN polymerase: C chc nng xc tc cho s tng hp phn t ARN t mch khun ca phn t AND theo chiu 5 3. 2.1.2.4. Cc Nuclease L enzyme thu phn cc lin kt phosphodieste gia cc nucleotit ca phn t axit nucleic. Gm 2 nhm: - Exonuclease: ct cc lin kt phosphodieste gia cc nucleotit ca phn t ADN t cc u ca phn t. - Endonuclease: ct cc lin kt phosphodieste gia cc nucleotit ca phn t AND ti cc v tr bn trong mt cch c hiu. v d: RE, S1 nuclease (ch ct si AND mch n), ADNse I (ct c si AND n v kp), 2.1.2.5. Cc enzyme sa i Alkaline phosphatase: Loi b nhm phosphat t u 5 ca phn t AND. Polynucleotide kinase: B sung nhm phosphat vo cc u 5 (c dng khi nh du ng v phng x nguyn t phospho vo phn t AND) Terminal deoxynucleotidyl transferase: b sung mt hay nhiu bp vo cc u 3 ca phn t AND. 2.1.3. Cc vector nhn dng ch yu c s dng trong cng ngh AND ti t hp 2.1.3.1. Khi nim Nhn dng gen: Qu trnh nhn bn cc on AND c ct thnh nhiu bn ng nht v sp xp chng thnh nhiu dng ring. Vic tch dng gen thng c thc hin thng qua t bo ch (thng l vi khun E.coli). Trong thc t, mt phn t AND bt k khng th t nhn ln trong t bo nu n khng c gn vi mt im khi u ti bn (ori), bi cc AND polymerase ch c chc nng gn kt cc bp t do trong mi trng vo u 3 OH ca bp trc n hoc im khi u ti bn ch khng c kh nng t khi u qu trnh ti bn bt k im no trn phn t ADN. Do , mun on gen no c nhn ln trong mt t bo bt k th chng ta phi gn on gen vi mt phn t AND c im khi u ti bn hay mt vector nhn dng. Vector nhn dng thc cht l mt phn t AND c gc ti bn v c th t nhn ln mt cch c lp vi b gen ca t bo ch. Cc vector nhn dng thng c s dng nht l plasmid, cosmid, bacteriophage, vic la chn loi vector nhn dng no tu thuc vo kch thc on gen cn nhn dng, nu on gen c kch thc nh hn 10kb th ngi ta thng chn plasmid nhn dng, nu on gen c kch thc 9 20kb th chn bacteriophage, nu t 33 37kb th chn cosmid, Yu cu ca vector nhn dng: (i). C im nhn bit ca mt hay mt s RE. Vector nhn dng l tng l ch c mt im nhn bit duy nht tng ng vi mi RE, v nu mt RE c nhiu im nhn bit trn mt vector th s dn n ri lon cho vic ct vector vi ng thi hai hay nhiu loi RE. (ii). C ch th di truyn chn lc (tnh khng khng sinh, tnh xc tc cho cc phn ng to mu,) c th xc nh c cc dng t bo no mang vector nhn dng c gn vi on gen quan tm. (iii). C im khi u ti bn (ori) m bo vector c th nhn ln mt cch c lp vi genom ca t bo sinh vt ch. 2.1.3.2. Mt s vector nhn dng c s dng ph bin hin nay A. Cc vector l plasmid Plasmid l cc phn t AND mch vng nh, nm ngoi nhim sc th ca t bo vi khun v c kh nng nhn ln mt cch c lp khng ph thuc vo s ti bn AND trong nhim sc th ca vi khun. Plasmid cn p ng c cc yu cu sau: (i) kch thc nh; (ii) c gen ch th cho php d dng pht hin, nhn bit plasmid trong t bo vi khun (thng l gen khng khng sinh hoc gen tng hp cht mu); (iii) c v tr nhn bit duy nht ca nhiu enzyme gii hn, (iiii) c im khi u ti bn. Mt s plasmid c s dng nhiu trong tch dng gen l plasmid pBR322, pUC18, pTZ57R/T, pBT...

S loi plasmid c trong t bo vi khun c th l 1 2 loi thm ch t 8 10 loi plasmid v lng bn sao ca mi loi plasmid trong t bo vi khun cng khc nhau, tu thuc vo loi plasmid (do tnh c hiu ca gc ti bn trong plasmid) v vi khun no. Tuy nhin, lng plasmid trong t bo thng dao ng trong khong 0,1 5% AND tng s. Cc plasmid trong t nhin c mt s nhc im nh: kch thc ln, cha nhiu gen khng cn thit cho s nhn dng cht lng cao v thiu cc gen quan trng nh gen ch th di truyn. Chnh v th, ngi ta thit k li cc plasmid theo nhng tiu chun mong mun. Mt s loi plasmid thng dng: (1) pBR322: - kch thc: 4361bp - gen ch th: gen khng Amp v Tetracylin - im nhn bit ca cc RE: BamHI, HinIII, SalI v PstI, EcoRI. - C mt gc ti bn, c hiu duy nht vi t bo E.coli (2) pBR325: - gen ch th: gen khng Amp , Tetracylin v Choramphenicol c to ra t pBR322 bng cch chn vo v tr EcoRI gen m ho tng hp Choramphenicol acetyltransferase. (3) Nhm plasmid pUC (4) Vector pBT (5) Vector pTZ57R/T i vi pUC, pTZ57R/T v pBT, v tr a nhn dng c thit k nm trong phn gen lacZ. Gen ny m ho enzyme galactosidase, enzyme ny s chuyn ho c cht X gal (khng mu) thnh cht X v galactose, cht X mang mu xanh chm. Bnh thng, nu lac-operon hot ng (plasmid t ng vng tr li), s c s to galactosidase nn cc khun lc mang cc plasmid ny s c mu xanh do c cht X-gal b enzyme phn gii to nn cht X c mu xanh, l nhng khun lc khng mong mun do n khng mang on gen quan tm. Nu lac-operon khng hot ng (do on gen quan tm hoc mt on gen bt k no c gn vo vector), s khng c s to thnh galactosidase v th cc khun lc mang cc plasmid ny s c mu trng, y c th l nhng khun lc cha on gen quan tm . B. Vector l phage Cc plasmid ch c th mang c cc on gen c kch thc nh (<10kb), nhn dng cc gen c kch thc ln hn th ta cn s dng cc phage. Cc phage c mt s u im so vi plasmid: (i) on AND chn vo c kch thc ln hn (9 20kb); (ii)Hiu qu chuyn np vo t bo ch cng cao hn do kh nng xm nhim ca phage cao hn. Cc loi phage c s dng hin nay: T4, M13, lambda. C. Vector l cosmid Cho php chuyn ti on gen c kch thc ln (33 45kb). Cosmid l s kt hp gia phage v plasmid, bao gm: u cos (u dnh) ca phage: bao bc ly AND v xm nhim vo t bo Gc ti bn ca plasmid: cosmid nhn ln c lp vi genom ca t bo vi khun. Do hu ht cc gen trong phage b ct b nn cosmid c th mang nhng on AND ngoi lai c kch thc ln. 2.2. Cc k thut chnh s dng trong phn tch ADN 2.2.1. K thut in di phn tch ADN Cc phn t ARN v ADN vi khi lng khc nhau c tch ra khi di chuyn trong in trng t cc m sang cc dng ca my in di. Vi nng agarose, in th v cng thch hp th c gii phn tch cc on ADN kch thc khc nhau. K thut in di cho php pht hin vt ADN khi nhum vi ethidium bromide. Ethidium l mt cht tng ng vi cc baz nn n xen k vo si ADN, di nh sng UV th pht quang v pht quang t l thun vi lng ADN. Chiu di bc sng ti u l 254nm nhng trnh lm t gy si ADN trong thc t ngi ta s dng bc sng 300nm. S chuyn ng ca ADN ph thuc vo 3 yu t:

- ln ca on ADN: nhng on ADN kch thc b chuyn ng nhanh hn v s chuyn ng c mi quan h logarit vi di on ADN. - Kiu hnh ca ADN: iu ny thng thy i vi plasmid dng siu xon, si vng, si thng. V vy tch v phn bit kch thc plasmid thng phi dng mt s enzym gii hn ct plasmid to ra dng mch thng. - Nng agarose. 2.2.2. K thut nhn dng gen Nhn dng gen l qu trnh nhn bn tng on AND t mt hn hp cc on AND vn l sn phm ca qu trnh ct bng RE hoc phn ng PCR. Qu trnh nhn dng gen bao gm cc bc: B1: Tch chit AND t mu nghin cu, to cc phn on AND quan tm bng cch ct bng RE hoc phn ng PCR. B2: Gn on ct vo mt vector nhn dng. B3: Bin np vector nhn dng mang gen quan tm vo t bo sinh vt ch nhn bn on gen quan tm. B4: Chn lc cc dng t bo sinh vt ch mang on gen quan tm. Hin nay ngi ta thng s dng PCR nhn dng gen, thc hin nhn dng bng phng php ny gm cc k thut sau: 2.2.2.1. K thut tch chit gen H gen ca sinh vt c th l ADN (thc vt, ng vt, vi khun, nm) hoc ARN (virus, th thc khun). V th k thut tch chit gen cng bao gm k thut tch chit ADN v k thut tch chit ARN. n nay, c nhiu phng php tch chit gen c a ra, tuy nhin tt c u theo nhng nguyn tc chung: Ph v t bo, gii phng cc thnh phn bn trong. Loi b protein v cc thnh phn khng mong mun khc. Thu gen. A. K thut tch chit ADN y l quy trnh c s dng ph bin trong k thut di truyn, c bit l trong vic xc nh a hnh di truyn mc phn t. Vi cc i tng sinh vt khc nhau (thc vt, ng vt, vi khun, ), quy trnh tch chit ADN l khc nhau. Quy trnh tch chit ADN thc vt theo phng php CTAB: 1) sn 5ml m chit 600C trong 30 pht. 2) Nghin 0,75g m l thc vt trong nit lng. 3) Ha tan mu nghin trong m chit, mu 650C trong 30 pht. Lc u, o nh 3 5 ln trong qu trnh . 4) B sung Chloroform : Isoamyl alcohol (24 :1), lc u. 5) Ly tm 13000 vng/pht trong 5 pht 40C. 6) Chuyn dch ni sang ng sch, b sung mt lng tng ng vi 2/3 th tch dch ni isopropanol hoc cn 100% c b sung natri acetat 3M. 7) mu -200C trong 3 - 4 gi, ly tm 13000 vng/pht trong 10 pht 40C. Loi dch. 8) Ra ADN bng cn 80%, ly tm 13000 vng/pht trong 5 pht 40C. 9) Lm kh ADN, ha tan trong dung dch TE 0,1X. 10) B sung RNase t nng cui cng l 100mg/ml, 370C trong 1 gi. 11) Cc bc tip theo tng t t bc 7 10. B. K thut tch chit ARN Hu ht gen sinh vt nhn chun u c cu trc gin on, bao gm cc on m ha (Exon) v on khng m ha (Intron) xen k nhau. V th khi xc nh trnh t m ha ca mt gen no phc v cho mc ch chuyn gen hoc biu hin gen, th ngi ta thng s dng phng php tch chit ARN tng s thu c thng tin v on gen ch bao gm nhng on m ha cho cc axit amin.

Vic tch chit ARN cn c ng dng trong nghin cu gen ca virus, th thc khun c h gen l ARN. Tch chit ARN tng s nhn chung cng ging nh tch chit ADN. Tuy nhin, dch chit sau khi lm sch protein c vi enzyme DNase thy phn ADN. Quy trnh tch chit ARN tng s theo b kit Trizol Reagents t thc vt: - Cn 100mg mu, nghin nhanh trong nit lng, m bo mu phi c nghin trit v trnh enzyme ARNse ct ARN. - Chuyn ngay mu vo ng eppendorf 2ml. - B sung Trizol Reagents, o u nhit phng (15 300C) trong 5 pht. - B sung mt lng Chloroform : Isoamyl (24 : 1), o u nhit phng trong 5 pht. - Ly tm 10000 v/p trong 15 pht. - Thu dch ni, b sung mt lng tng ng Isopropanol c lm lnh n 40C, lc nh ng ARN kt ta. - nhit phng trong 10 pht, ly tm 10000 v/p trong 15 pht. - Loi b dch ni, ra ta ARN bng cn 70% pha trong DEPC 0,01%. - Ly tm 6000 v/p trong 5 pht, loi dch. - Lm kh ARN. - Pha long ARN trong H2O x l DEPC 0,01%. - nhit 550C trong 5 pht ARN tan ht. Do ARN d b RNase ct nn tt c cc dng c u phi x l DEPC 0,01% v kh trng trc khi s dng. 2.2.2.2. K thut tng hp cADN (Comprementary DNA) ngn hng cADN Vi nhng sinh vt c h gen l ARN, tch dng mt on gen quan tm, ngi ta khng bt u vi ARN m bt u vi cc trnh t ADN b sung (cADN) vi cc trnh t ARN genom. Mt khc, ADN sinh vt nhn chun bao gm cc on exon v intron xen k nhau nn trong qu trnh sao m phi c qu trnh loi b cc intron to mARN hon chnh, c th tham gia sinh tng hp protein. B my enzyme chu trch nhim ct b cc intron ny ch c sinh vt nhn chun m khng c vi sinh vt nn vic chuyn np mt gen c cha c phn intron vo vi khun l khng hp l. khc phc tnh trng ny, ngi ta cng s dng cc trnh t ADN b sung vi mARN ca gen quan tm. Qu trnh tng hp cc cADN t mARN c thc hin nh enzyme sao m ngc (Reverse transcriptase). Loi enzyme ny c pht hin trong nghin cu qu trnh ti bn b gen ca virus c genom l ARN. Nh vy, cADN l cc phn t ADN c tng hp t khun mARN qua qu trnh sao chp ngc, n c lu gi trong ngn hng d liu cADN. Nguyn l ca qu trnh ny l s dng enzyme sao m ngc tng hp nn si cADN th nht t ARN. Ty theo mc ch ca th nghim m cc loi ARN c s dng l khc nhau. Nu mun nhn mt on gen ca sinh vt bc cao m khng c intron th ngi ta thng tng hp cADN t mARN vi mi oligo(dT)s. Nu l sinh vt bc thp nh vi khun, virus... ngi ta c th tng hp cADN t ARN tng s vi mi ngu nhin hoc mi c hiu cho gen cn nhn ln. Khi mi gn b sung vi si ARN khun, enzyme sao m ngc s xc tc cho qu trnh tng hp cADN t cc nucleotide t do c trong thnh phn phn ng mt nhit thch hp. Phn ng tng hp cADN ch xy ra trong mt chu k, theo l thuyt sau khi kt thc phn ng ta s thu c lng cADN tng ng vi lng ARN khun ban u. tng hiu qu ca qu trnh tng hp, lng mi c cho vo ln hn rt nhiu lng ARN khun v cht km hm ARNse cng c a vo bo v ARN khi tc ng ca RNase. 2.2.2.3. PCR (Polymerase chain reaction) A. nh ngha Genom ca sinh vt bc cao cha mt lng gen rt ln ( genom ca ng vt bc cao cha khong 100.000 gen), v vy, vic tch chit v nghin cu mt gen c th t khi lng gen khng l v phc tp ny l rt kh khn, phc tp. Nm 1985, Karl Mullis v cs m t phng php PCR, phng php ny s gip cho chng ta to ra c mt lng

ln bn sao ca on ADN quan tm, v th vic tch chit v nghin cu mt gen c th trong genom ca sinh vt tr nn d dng hn. PCR l k thut in vitro tng i n gin cho php nhn nhanh mt s lng khng hn ch nguyn bn mt on ADN nht nh trong mt khong thi gian ngn, nh s xc tc ca ADN polymerase. B. Nguyn tc thc hin Nhn bn ADN trong t bo c th c chia lm 3 giai on: - Giai on dui xon v phn tch 2 mch n ca phn t ADN. - Giai on gn mi vo trnh t nucleotit b sung trn 2 mch ca ADN. - Giai on ko di 2 mch ADN b sung, kt thc nhn bn. Phn ng PCR cng gm c 3 bc v c lp i lp li nhiu ln. PCR cng c thc hin theo nguyn tc b sung v nguyn tc gi li mt na nh c ch ti bn ADN trong t bo. Qu trnh nhn bn bng ADN polymerase bao gm 3 bc c lp li nhiu ln: - Bin tnh ADN t dng si kp thnh dng si n bng cch nng cao nhit ln 94-950C trong khong thi gian 30 giy n 1 pht. - Gn mi vi on ADN cn khuych i thng qua h nhit xung 40-600C, trong khong thi gian 30 giy n 1 pht, ph thuc vo di v t l (G + C)/(A + T) ca on mi. - Phn ng polymerase tng hp phn t ADN ko di t u 3' - OH ca mi. Hai phn t ADN mi c tng hp theo nguyn tc b sung t hai u ca phn t ADN lm khun. Thi gian ko di t 30 giy n vi pht. trnh hin tng bt cp khng c th, phn ng tng hp nn c thc hin 720C. Nh vy, sau mi chu k phn ng lng ADN cn nhn bn s tng gp i, vi N si khun ban u sau n chu k s c Nx2n si ADN c nhn ln. thc hin c phn ng PCR, chng ta cn c nhng thnh phn g? Ti sao li gy bin tnh ADN bng cch nng nhit ln n 94 950C? Chiu tng hp ca phn ng? C. Thnh phn phn ng 1. ADN polymerase ADN polymerase l nhng enzyme c chc nng gn thm cc nucleotide vo u 3'-OH ca trnh t mi cng nh trnh t ca on DNA. Ngoi ra, polymerase cn c kh nng sa sai trong qu trnh ko di chui DNA nh hot tnh exonuclease 5 3. Trc y, ngi ta s dng cc ADN polymerase bnh thng (chit t E.coli) trong phn ng PCR, nhng hiu qu khng cao do phi b sung mt lng enzyme nht nh trong mi chu k phn ng. Ngy nay, nh pht hin ra ADN polymerase chu nhit cao (taq polymerase tch t vi khun Thermophilus aquaticus), vic thc hin phn ng ny tr nn n gin v t hiu qu cao hn. Taq polymerase? Taq polymerase l ADN polymerase chu nhit cao, c tch t vi khun sui nc nng, c hot tnh ti u 720C, nhng n c th bn ti 940C, trong 1 giy taq polymerase gn c 75 nu vo chui polynucleotit 2. ADN khun Lng ADN khun c th ch l 1 phn t ADN th phn ng PCR vn cho kt qu dng tnh. Khc vi vic nhn gen bng phng php to dng, phn ng PCR khng i hi lng ADN khun phi ln, thm ch khi ch c mt phn t ADN khun th phn ng PCR vn c th din ra. iu ny lm cho PCR d b sai nu mu nghin cu b nhim vi ADN khc. u th vi mu l khng cn tinh sch cao. C th tin hnh PCR vi cc mu mu, cc mu kho c hoc vi khun b hp kh trng,... Tuy nhin, trong nhiu trng hp cn chun b mu tt phn ng PCR cho kt qu chnh xc. 3. Cc mi (primer)

Tt c cc ADN polymerase khi hot ng tng hp mt mch ADN mi t mch khun u cn s hin din ca nhng mi c hiu. Mi l nhng on ADN ngn c kh nng bt cp b sung vi mt u ca mch khun. ADN polymerase c chc nng ni thm cc nucleotide vo trnh t mi hnh thnh mch ADN mi. Cc primer c vai tr rt quan trng quyt nh n mc thnh cng ca phn ng PCR. Mi cn m bo 3 tiu chun sau: + Kch thc hp l (khong 6 30b) + Mi khng c bt cp b sung vi nhau + Trnh c t l C+G/A+T cao Thi gian gn mi vo 2 mch ca ADN ph thuc vo nhng yu t no? 4. dNTP - nucleotide triphosphate dNTP gm c dATP, dTTP, dGTP v dCTP. y l nguyn liu tng hp cc chui polinucleotide trong phn t ADN. 5. Dung dch m thch hp v MgCl2 Dung dch m v MgCl2 c vai tr g trong phn ng PCR? Dung dch m lm cho pH mi trng phn ng n nh v ph hp vi pH ti u ca cc enzyme tham gia. Mg++ l ion cn thit cc ADN polymerase hot ng ti u. D. Mt s c im ca PCR 1. PCR cho php nhn mt on gen nht nh (gen ch) nh cp mi c hiu vi gen . on gen ch chnh l on nm gia v tr bt cp ca 2 cp mi trn phn t ADN. S dng PCR n nhn gen ch mt 2 3h trong khi , nu s dng h thng t bo (E.coli) th phi mt 1 vi ngy. 2. Taq polymerase khng c kh nng sa cha nhng kt cp sai trong qu trnh ko di chui. Theo tnh ton l thuyt, s nhm ln xy ra y c tn s xut hin l 1 trn 2x10 4 nucleotide (Tn s bt cp sai cc nucleotit trong ti bn ADN t nhin l 1/106). Tuy nhin nhng sai st ny khng nghim trng v trong PCR, nhiu chu k nhn xy ra trn mt on ADN ging nhau, nn s ADN c 1 nucleotide sai lch l khng ln. 3. PCR c nhy cao, lng ADN khun c th ch l mt phn t. Do , kt qu nhn bn ADN d b nhiu khi ngun nhn c ln d ch mt phn t ADN. 4. Mu ADN a vo chy phn ng PCR khng cn c tinh sch cao, v th khng nht thit phi tin hnh PCR vi cc mu ADN tinh sch m c th s dng cc mu mu, tc, cc t bo vi khun,... y l mt trong cc u th lm cho PCR c ng dng rng ri trong k thut di truyn. E. Gi tr s dng Do nhng c tnh u vit nh: thi gian thc hin nhanh, n gin, t tn km, tinh sch ca mu khng cn cao m ngay t khi ra i, PCR c ng dng rt rng ri trong cc lnh vc khoa hc cng nh trong i sng x hi. - Trong k thut di truyn: PCR gip cho vic xc nh trnh t nucleotide ca on nhn, v th n c ng dng trong vic xc nh c im cu trc ca gen, cc thnh phn ca gen, t c ng dng trong vic pht hin ra cc t bin gen.,... T nhng mu xng, lng,... cn li ca cc ng vt cht, ngi ta c th xc nh loi, gii tnh,.... ca n. - Trong y hc: PCR c hiu qu ng dng cao trong vic chn on s ly nhim cc bnh virus, vi khun, nm vo c th sinh vt ni chung v con ngi ni ring. Mt khc, n cn gip cho vic chn on sm cc bnh ung th, cc bnh di truyn, xc nh gii tnh,... Nh c PCR v k thut nhn dng gen m con ngi c th sn xut mt lng ln insulin, vitamin, axit amin, cc loi khng sinh,... - Trong khoa hc hnh s: PCR gip cho vic pht hin ti phm t nhng mu sinh phm ca chng li trn hin trng, cho php xc nh quan h huyt thng,... - Trong chn ging: PCR l mt trong cc ch th phn t c hiu qu ng dng cao trong vic chn lc cc kiu gen, xc nh c im di truyn phn t ca con lai F1, phn lp v chn lc tnh trng,...

Chuyn gen khng su, thuc tr c, khng virus, chuyn gen to ging hoa c nhiu kiu v mu sc mi,... Tm li, sc mnh ca PCR kt hp vi cc k thut khc ca to dng phn t gip sinh hc xm nhp vo nhiu lnh vc khoa hc m trc y kh vi ti. 2.2.2.4. K thut bin np plasmid vo E.coli v chn dng t bo Plasmid sau khi c tin hnh phn ng lai vi on gen quan tm cn phi c a vo h thng t bo sng nhn bn on gen quan tm thng qua h thng t bo. Nm 1928, Griffith m t qu trnh bin np vi khun trong th nghim ni ting ca ng v nguyn l bin np. Tuy nhin, khng phi tt c cc vi khun u c kh nng bin np. bin np c hiu qu, cc t bo vi khun cn tr nn kh bin. t c iu ny, ngi ta gi cc t bo vi khun trong dung dch CaCl2 trong . Khi cc t bo tr nn kh bin v c kh nng cho php cc plasmid i qua mng vo bn trong t bo. Vic bin np plasmid vo t bo kh bin c thc hin bng cch: - Trn DNA plasmid vi cc t bo. - 40C trong 20 - 30 pht. - Sc nhit 420C trong 1 pht plasmid c th xm nhp vo t bo. - Nui lc vi khun trong mi trng LB 370C trong vng 60 n 90 pht. - Cc t bo vi khun c cy tri trn mi trng chn lc c khng sinh v c cht to mu nhn ln cc t bo c plasmid, mi t bo s pht trin thnh mt dng ring bit. Trong qu trnh bin np, ch c mt lng nh t bo kh bin c bin np. Bin np l mt k thut quan trng c th to ra khong 109 cc t bo bin np khi s dng 1 g DNA Sau khi bin np thnh cng, chn c nhng dng t bo mang gen quan tm, cn phi tin hnh chn lc dng t bo. Vic chn chn dng vi khun ti t hp c th thc hin theo hai cch: chn lc theo phng php khng khng sinh (kanamycin hoc ampicillin) nhm loi tr cc t bo vi khun khng c bin np v cc loi vi khun nhim tp khc v chn lc theo phn ng vi c cht (X-gal/IPTG) nhm loi tr nhng vi khun c mang vector nhng khng c on gen c chn vo. khng nh chnh xc dng t bo no mang on gen quan tm, c th thc hin theo 2 cch: (i) tin hnh phn ng PCR t nhng khun lc nghi vn; (ii) tin hnh phn ng ct enzyme ca cc khun lc nghi vn. 2.2.3. K thut xc nh trnh t ADN xc nh c trnh t nucleotit ca mt on phn t ADN no , trc ht cn phi c qu trnh tinh sch on gen . Thng thng, ngi ta tin hnh qu trnh tinh sch gen quan tm trc khi xc nh trnh t bng cch tch plasmid ca mt dng khun lc c xc nh chnh xc l c mang on gen quan tm v tinh sch plasmid . C nhiu phng php xc nh trnh t ADN 2.2.4. Cc k thut lai axit nucleic Nguyn l: Da vo tng tc gia cc cp baz theo nguyn tc b sung, on polynucleotit n c cu trc b sung vi nhau hon ton c th bt cp vi nhau to ra phn t ADN lai. iu kin: bin tnh ADN cn nhit cao hoc cc cht gy bin tnh (NaOH, formaldehyt); mu d phi c nh du phng x. 2.2.4.1. Lai Southern Blot Phng php ny c Edwin M. Southerm xy dng v th nghim thnh cng nm 1975. Phng php ny cho php xc nh c s c mt ca mt on gen no trong genom ca t bo sinh vt. Thc cht, y l phng php lai phn t ADN vi cc ADN mu d c nh du phng x P32 hoc nh du bng cht to mu. Trc ht, ADN c ct bng enzyme hn ch, sau chy in di trn gel agarose, b gy bin tnh (thng dng NaOH) ri thm truyn ln mng lai nitrocellulose, cui cng lai vi mu d ADN tng ng ( c chun b trc) v s dng k thut phng x t ghi, cc phn ng lai s c hin th trn phim thnh tng bng vch ngang. 2.2.4.2. Lai Northern

Phng php lai Northerm l lai gia ARN t bo vi mu d (ADN hoc ARN) c nh du. Phng php ny cho php ta xc nh c s c mt ca mARN trong t bo (xc nh c s hot ng ca gen quan tm trong t bo). 2.2.4.3. Lai Westhern L php lai gia protein t bo vi cc protein mu d c nh du. Phng php ny c thc hin da trn nguyn tc phn ng min dch khng nguyn khng th, trong cc protein t bo l cc khng nguyn, mu d l khng th. Ti nay c nhiu ci tin phng php ny hnh thnh nhiu k thut phi hp nh Southwestern, pht hin bng protein bng ADN nh du,... Lai Western cho php ta xc nh c s biu hin ca gen trong t bo sinh vt ch to ra cc protein quan tm. 3 phng php lai trn c ng dng rt rng ri trong cng ngh gen, c bit l trong nghin cu to sinh vt chuyn gen. Lai Southern cho php xc nh s c mt ca gen chuyn trong t bo sinh vt ch (gen chuyn c gn vi genom ca t bo sinh vt ch hay cha? S lng bn gen c gn?) tuy nhin k c khi xc nh c s c mt ca gen chuyn trong genom t bo sinh vt ch th cng khng c ngha l chc chn gen s hot ng. Chnh v th, lai Northern cho php xc nh s hot ng ca gen chuyn mc to ra mARN v lai Western cho php xc nh c s biu hin ca gen chuyn to ra sn phm mong mun l cc protein. Trong to cy chuyn gen, tu thuc vo mc ch chuyn gen m khi kim tra cy chuyn gen cn quyt nh tin hnh nhng php lai no k trn. 2.2.5. Cc phng php xc nh tnh a hnh ADN 2.2.5.1. K thut nh gi a dng v chiu di ca cc phn on ADN c ct bng RE (Restriction Fragment Length Polymorphism RFLP) T bo sinh vt c kh nng nhn bn ton b genom ca chng vi tc v tnh chnh xc cao. Tuy nhin, trong qu trnh nhn bn ADN, c kh nhiu khu hot ng sai lch gy nn nhng thay i trong phn t ADN, c th l thay i mt hay mt vi cp baz hay nhng thay i chuyn, o, lp, mt on,.... Kt qu l kh c th c 2 c th c cc chui ADN ging nhau mt cch tuyt i, v chnh iu to nn s a dng rt ln v mt di truyn ca sinh gii. S dng tch dng v xc nh trnh t nucleotit ca ton b genom sinh vt nh gi tnh a dng di truyn hay tnh bin d di truyn ca sinh vt th qu tn km v i hi nhiu thi gian. hn ch c nhng nhc im ny, c th tin hnh xc nh mc bin d ADN bng cch s dng cc RE. Cc RE s ct cc phn t ADN thnh nhng on c chiu di khc nhau, s lng phn on v chiu di ca cc phn on phn nh s lng cc v tr hn ch c trn chui ADN m ta quan tm. T , nu chn c t hp ADN/RE ta c th xc nh c 2.2.5.2. Phng php nhn cc on ADN a hnh ngu nhin (Random Amplified polymorphic DNA RAPD) Mt trong nhng hn ch ca PCR l n i hi phi c s bit trc trnh t (hay t nht mt phn) ca on ADN cn nhn, t c th thit k c 2 cp mi c hiu. V th, mc a dng ca on nhn kh c m rng. Nm 1991, Gustavo Caetano Anoless v cs pht hin ra hin tng nhn cc on ADN bng PCR ch vi mt on mi bt k. T ra i k thut nhn cc on ADN a hnh ngu nhin (RAPD). RAPD l phn ng polymerase dy truyn nhm nhn cc on ADN ring bit, ngu nhin nm ri rc genom vi s s dng mt loi mi. y, cc on ADN nhn bn khng cn bit trc trnh t nucleotide. Trong RAPD, mi thng c di 7 10bs. Trn ADN genom, c nhiu im tng hp vi on mi v th c nhiu on ADN khc nhau c nhn ln. RAPD c ng dng nhiu trong phn tch kiu gen, chn lc c th theo ch th phn t. RAPD khc PCR nhng im sau: 1. S chu k nhiu hn (RAPD: 40 45, PCR: 25 32 ch k). 2. Mi RAPD l mi ngu nhin cn mi tin hnh PCR l mi c hiu. 3. Trong RAPD ch c 1 mi, trong PCR c 2 mi (mi xui v mi ngc). 4. Mi RAPD thng di 7 10 bp, cn mi trong PCR thng di 6 30bp).

5. S phn on ADN c nhn ln trong RAPD nhiu hn trong PCR rt nhiu. 6. RAPD thng c s dng trong nghin cu a hnh ADN cn PCR thng c ng dng trong phn lp gen, chuyn gen,...