CC K THUT PCR V NG DNG

K THUT RFLP (Restriction Fragment Length Polymorphisms) I. Gii thiu K thut RFLP l k thut nghin cu tnh a hnh chiu di ca cc phn on DNA da trn im ct cc enzim gii hn (Restriction Enzyme, RE). Khi DNA vi enzim gii hn dung dch m thch hp pH, nhit thch hp s s to ra nhng phn on DNA vi kch thc khc nhau, t lp nn cc bn gen. K thut ny c s dng ph bin t thp nin 80 n nay. II. Nguyn tc chung Nguyn tc ca k thut ny da trn c hiu ca cc enzim ct gii hn (restriction enzim-RE) i vi v tr nhn bit ca chng trn DNA b gen. S khc bit v tr ct gia hai c th s to ra nhng phn on ct khc nhau. III. Cc enzim gii hn (RE) 1. Gii thiu Enzim gii hn c Werner Arber tm thy vi khun vo nm 1962. ng cho rng cc RE c kh nng rt c bit l kh nng nhn bit DNA ch v DNA l. Enzim ny hn ch s nhn ln ca DNA l khi chng xm nhp vo t bo vi khun bng cch ct chng ra thnh tng on mt cch c hiu, v th ng gi l restriction c ngha l hn ch. Vi pht minh ny ng cng cc cng s (Daniel Nathans v Hamilton O. Smith) ginh gii thng Nobel vo nm 1978. Cc RE hp thnh h thng bo v t bo s hch (procaryote), mt cu hi t ra l v sao cc RE ch ct DNA ca cc phage m khng ct DNA ca t bo vi khun? Cu tr li l nh Methylase y l enzim gip vi khun c kh nng ny, chnh enzim ny chu trch nhim gn nhm metyl (CH3) vo cc nucleotides v tr ct ca cc RE trn DNA ca vi khun v th bo v DNA ca vi khun khi s phn ct ca RE. Tuy vy i lc methylase li gn nhm c nhm metyl vo chnh DNA ca phage, iu ny gii thch v sao i vi cc dng vi khun khng phage vn c cc t bo b phn hy. Enzim ct gii hn u tin c phn lp vo nm 1968, cho n nay c khong 3.400 RE c khm ph. Trong s ny c khong 540 RE c thng mi ha.

2. Tn gi ca enzim ct gii hn

Ch u vit hoa l ch u tn vi khun t RE c ly trch. Hai ch k khng vit hoa tng ng vi tn loi ca vi khun. C 3 ch ny u vit in nghing. K n l ch s la m ch th t RE c pht hin trong trng hp nhiu RE c tm thy trong cng mt vi khun. i khi cn c thm ch vit hoa (vit thng) ch chng vi khun s dng VD : Escherichia chi coli loi Ry13 chng

EcoRI: l enzym u tin tm thy trn vi khun E. coli EcoRV: enzym th 5 tm thy trn vi khun E.coli 3. Cc loi enzim ct gii hn Loi I: khi enzym nhn bit trnh t n s di chuyn trn phn t DNA cch 1000-5000 nu v gii phng vi chc nu. Loi II: enzym nhn bit trnh t v ct ngay v tr . Loi III: enzym nhn bit trnh t v ct DNA v tr cch 20 nu. III. Quy trnh thc hin RFLP bao gm cc bc: + + + + Tch chit v tinh sch DNA Ct cc mu DNA cn phn tch bi RE Phn tch DNA trn gel agarose Southern Blotting

Chuyn DNA sang mng nylon Chn on d (probes), nh du on d Lai ghp gen (Hybridization) Lp bn gen (phn tch a hnh) 1. Trch DNA

1.

a.

Nguyn tc: Thnh t bo b ph v bng bin php c hc v cc

cht ty mnh. DNA c gii phng v lm sch tp cht Rnase. DNA c kt ta trong Ethanol 100% v thu li nh ly tm.

2.

b.

Quy trnh: Thc hin theo qui trnh c m t bi Rogers v

Bendich (1988) (qui trnh CTAB) gm cc bc sau: - Ly l ca i tng cn nghin cu. - Kh trng b mt l bng cn 70%, sau dng ko v kp ( c kh trng) ct l thnh tng mnh nh ri cho ring vo cc tup (mi mu ring mt tup khong 0,1g). - Ngm cc tup v dng c nghin mu vo nit lng trong 10 pht. - Cho mu vo my nghin, c tn s 27ln/giy, lp li khong 3 ln. - Cho thm 1.000l Extraction buffer (10ml EB +7l -Mercaptoethanol). - Cho thm 50l SDS 10%. - trong nc 65oC trong 30 pht. Sau 5 pht tr mt mu mt ln. - Ly tm 13.000 rpm trong 10 pht. - Ly 800l dch trong trn cho vo tup mi (b phn cn) - Cho 800l Isopropanol vo mi tup, lc u. - mu -20oC trong 2 gi. - Ly tm 13.000rpm trong 10 pht, b nc, ly cn. - Cho thm 400l TE 0,1 vo, thm 10l RNase, 20 pht 37oC. - Cho 400l CTAB vo, 15 pht 65oC. - Cho 800l Chloroform/Isoamylalcohol, o nh (12ml Chloroform: 0,5ml Isoamylalcohol) - Ly tm 13.000rpm trong 5 pht. - Ly 700l phn trong chuyn sang tup mi. - Cho 1,4ml Ethanol 96% lm lnh vo, lc nh, 15 pht nhit phng. - Ly tm 13.000rpm trong 10 pht, b nc, ly cn. - Cho 700l Ethanol 70% lm lnh vo, lc nh. - Ly tm 13.000rpm trong 10 pht, b nc, ly cn.

- Lp li ln na vi Ethanol 70% lm lnh, thm ming tup trn giy. - Sy chn khng 45oC trong 10 pht. - Cho 200l TE 0,1 vo, tr mu -20oC. c. Xc nh nng DNA bng phng php o quang ph Nguyn tc: - Da vo s hp th mnh nh sng t ngoi bc sng 260 nm v 280 nm ca cc baz purin v pyrimidin. - n v OD260 nm tng ng nng 50mg/ml cho mt dung dch DNA si i. Do nng [DNA] DNA trong = mu c tnh x 50 theo x cng thc pha sau: long (mg/ml) OD260nm

- kim tra tinh sch ca dung dch c th o thm gi tr OD 280nm. - Dung dch axit nucleic c xem l sch (khng ln protein) khi t s : OD260nm/OD280nm nm trong khng 1.8 2.

2. Dng enzim gii hn ct DNA Mt s quy tc khi thc hin phn ng ct bng RE Nng NaCl ca dung dch m, nu phi s dng nhiu RE c nng NaCl ca dung dch m khc nhau th nn ct vi RE c nng NaCl thp trc ri n RE cn NaCl nng cao hn. RE c bo qun trong dung dch m c 50% glycerol -200C, nn thm vo phn ng sau cng, thi gian bn ngoi cng ngn cng tt. Th tch phn ng cng nh cng c hiu qu v RE v DNA tip xc vi nhau cng tt tuy nhin phi bo m th tch RE khng qu 1/10 th tch phn ng v glycerol nhiu s gy c ch hot ng ca RE.

3. Southern Blot a. Chuyn DNA sang mng nylon - Ct DNA thnh cc on nh bng enzim gii hn thch hp. - in di sn phm ct trn gel Agarose tch cc on DNA theo kch thc. - Lm bin tnh DNA, khi DNA cn trn gel bng dung dch kim.

V d: c th nhng DNA vo trong dung dch NaOH 0.5M : NaCl 1.5M, DNA si kp s c tch thnh DNA si n. - Ch DNA si n mi c th chuyn ln mng lai.Chuyn DNA bin tnh ln mng lai. - Mng lai c s dng l mng nitrocellulose. Ngi ta cng c th s dng mng nylon. Mng nitrocellulose in hnh c kh nng tip nhn 100g DNA/cm2, trong khi mng nylon c kh nng tip nhn 500g DNA/cm2. Mt khc mng nylon c kh nng gi DNA chc hn v t t gy hn. Vic chuyn DNA thng c tin hnh bng hot tnh mao dn trong khong vi gi hoc c th dng mt thit b thm chn khng. Nu dng thit b thm chn khng th s nhanh hn, ch mt khong mt gi. Trong qu trnh chuyn, v tr cc on DNA vn c gi nguyn khng thay i.

Vat nang Tam knh giay tham Mang lai Gel Giay tham day Dung dch chuyen
Acid nucleic c chuyn ln mng lai nh lc mao dn.

b. Cch chn on d (probes) Probe (on d) l mt si nucleic acid (hoc ribonucleic acid ) c trnh t xc nh v c nh du bng cc phng php khc nhau dng nhn din cc on nucleic acid khc c trnh t b sung vi n. Qu trnh ny da trn nguyn tc bin tnh v hi tnh ca phn t DNA, c gi l lai phn t DNA . Cc phng php c in c s dng probe l Northern Blot (nhn din bn RNA phin m ), Southern Blot (nhn din cc phin bn ca gene ). Cng ngh DNA chip v cDNA micro-arrays c thit k da trn nguyn tc s dng cc probe ca cc phng php lai phn t nhng cho php nghin cu ng lot hng nghn gene khc nhau.

Probe c th c thit k da trn nhng trnh t c sn tu vo mc ch ca tng th

nghim v cc thng tin c. + Da trn trnh t genome ca i tng nghin cu nhn din cc bn sao ca gen

hoc sn phm RNA ca gen. + Da trn trnh t genome ca cc sinh vt c quan h gn gi vi i tng nghin cu

nhm tm kim gen chc nng c bo tn qua qu trnh tin ho. + Da trn trnh t amino acid ca protein l sn phm ca gene. T tm kim trnh t

trn vn ca gene m ho cho protein trn genome. Trong qu trnh thit k on d cn bo m cc yu t sau: 1) tnh c trng ca probe, 2) khng c hin tng lai cho gia cc probe hoc to cu trc khng gian ni ti trong probe, 3) gi tr nhit bin tnh (Tm) phi ph hp khi thc hin php lai nhiu probe mt lc.

c. Cch ghi nhn on d cho phng php lai Southern

Cch ghi phng x

Thng dng: 3 H, 32P, 33P, S 35 Mu d c nh du trong mt phn ng sao chp DNA nh mt dNTP mang cht ng v phng x Xem kt qu: phng x t ghi (autoradiograph)

Cch ghi hunh quang

Mu d c nh du trong mt phn ng sao chp DNA nh mt dNTP mang cht pht hunh quang Xem kt qu: my o hunh quang

u im v khuyt im ca k thut RFLP - RFLP c u im l marker ng tri cho php phn bit c c th ng hp v d hp. Do kch thc DNA kho st trong RFLP ln v vy s lng du phn t (marker) to ra nhiu p ng nhu cu nghin cu.

- Tuy nhin do qui trnh thc hin phc tp, nguy him i vi sc kho ngi nghin cu (s dng phng x nh du), v DNA yu cu c cht lng cao lm hn ch vic s dng k thut ny. Hn na hin nay, nc ta nh nc cha cho php nhp cc cht phng x. - Cng vi s pht trin k thut PCR, k thut RFLP tr nn n gin hn. Mt cp mi oligonucleotide c th dng khuch i mt vng DNA cn kho st, sau on DNA c khuch i c ct bng cc RE, in di v phn tch trn gel nhum ethidium bromide hoc bc. PCR-RFLP b qua bc lai nn gi thnh r hn v t nguy him hn phng php RFLP.

IV. ng dng: - Trn Thanh Mn s dng k tht PCR-RFLP nghin cu a dng sinh hc cc ging bi (Citrus maxima (Burm.) Merr.) Vit Nam. Thc hin phn ng PCR vi 2 cp mi ITS 1 v ITS 2; ITS 1 v ITS 4. Cc sn phm PCR ca tng cp mi c ct bng 6 enzim gii hn SmaI, EcoRI, HindIII, PstI, BamHI, KpnI. Da vo kt qu thu c ta xc nh c mi tng quan di truyn ca cc ging cy ny. Bc s L Nht Minh, Phan L Thanh Hng v L Th Kim Tuyn s dng k

thut PCR-RFLP xc nh mt s vi khun l cn nguyn chnh gy vim mng no tr em. K thut PCR s dng mt cp mi chung cho php nhn ln mt on DNA c kch thc 996bp trn gen m ha cho yu t 16S ribosom ca 11 loi vi khun, sau sn phm PCR c ct bng enzim Hae III kt qu phn bit c 11 loi vi khun chun khc nhau v xc nh chnh xc 9 loi vi khun l cn nguyn chnh gy vim mng no tr em.(Trch Hi ngh Khoa hc 2006 Vin Pasteur TP.HCM) K THUT PCR (Polymerase Chain Reaction) I. Gii thiu PCR l ch vit tt ca cm t Polymerase Chain Reaction (Phn ng chui trng hp phn ng khuch i gen). y l k thut ca sinh hc phn t cho php nhn bn mt on DNA mong mun t h gen DNA ca c th sinh vt thnh nhiu bn sao, k thut ny c nh khoa hc ngi M Kary Mullis v cng s pht minh nm 1985 ti cng ty Cetus; mc d nguyn tc ny c m t chi tit trc mt thp nin bi Khorana v ctv., (Kleppe v ctv., 1971; Panet vctv., 1974), v c gii thiu ln u tin ti Hi tho ln th 51 Cold Spring Harbor vo nm 1986 v ng nhn c gii thng Nobel Ho sinh hc vo nm 1993. K t to nn mt tc ng to ln i vi cc nghin cu sinh hc trn ton th gii.

II. Nguyn l Nguyn l nhn gen trong t bo: DNA l vt cht di truyn ca c th sng quy nh ln c tnh ca c th (ngoi tr mt s loi virus c vt cht di truyn l RNA). i vi nhng sinh vt c cu trc t bo nhn chun, cc phn t DNA tn ti di dng kt hp vi protein thnh cc nhim sc th trong nhn t bo. Ngoi ra, ti cc c quan t nh ty th v lp th ( thc vt) cng cha DNA dng plasmid. Cc phn t DNA nhng t bo hot ng bnh thng ch c nhn ln trong qu trnh phn bo nguyn nhim. thc hin c qu trnh ny, i hi phi c mt enzim DNA polymerase, s tham gia ca cc on mi c trnh t b sung gn vo vo si DNA khun v cc dNTPs lm ngun cung cp nucleotit. Trong qu trnh phn bo nguyn nhim, cc si DNA kp c m xun tch thnh cc si DNA si n. Di tc dng ca enzim DNA polymerase, qu trnh tng hp DNA c xy ra theo cch gn ln lt cc nucleotit vo on mi ko di chui theo nguyn tc b sung vi DNA khun. Nguyn l ca k thut PCR K thut tng hp DNA ngoi c th cng tun th nhng nguyn tc c bn ca sao chp DNA trong c th nhng c s khc bit: + Dng nhit cao tho xon thay cho enzim helicase. + Kt hp vi enzim DNA polymerase chu nhit tng hp DNA mi trong mi trng thch hp. + H thng iu nhit thch hp cng vi cc on mi c thit k chuyn bit, ch ng. Phng php PCR cho php tng hp rt nhanh v chnh xc tng on DNA ring bit. y thc s l phng php hin i v thun tin cho vic xc nh s c mt ca mt gen no trong t bo vi chnh xc cao. Phng php ny da trn s khm ph hot tnh sinh hc nhit cao ca DNA polymerase c tm thy trong cc sinh vt a nhit (vi khun sng trong cc sui nc nng). Phn ln cc DNA polymerase ch lm vic nhit thp. Nhng nhit thp, DNA xon cht v vy DNA polymerase khng c nhiu kh nng lm bin tnh phn ln cc phn ca phn t. Nhng cc polymerase chu nhit ny hot ng nhit rt cao, c th ln n 1000C. nhit ny DNA s b bin tnh (DNA dng xon s dui ra v dng thng). Mt phn ng PCR l mt chui nhiu chu k ni tip nhau, mi chu k gm ba giai on: giai on bin tnh, giai on bt cp, giai on ko di.

III. My PCR Trc y, khi cha c my chu k nhit, thc hin c phn ng PCR cc nh khoa hc gp 2 tr ngi ln: + + Lc u enzim Klenow polymerase c s dng b phn hy 950C nn phi thm thay i chu k nhit cc nh khoa hc phi s dng 3 water baths 3 nhit khc

enzim vo ng nghim sau mi chu k giai on phn tch chui DNA. nhau. Khi m v thay i 30 n 35 chu k nhit kh phin phc, d gy sai st. Vic tm ra enzim Taq polymerase v my PCR t ng thay i cc chu k nhit lm cho k thut ny pht trin nhanh v c ng dng rng ri trong nhiu lnh vc nghin cu (McPherson v ctv., 1992). Mt cch tng qut, my PCR gm cc b phn chnh nh sau : + Mt bn phm n gin lp cc chng trnh chu k nhit v ra cc mnh lnh my thc hin. + Mt b vi x l ghi nh cc chng trnh np vo my, thc hin cc mnh lnh

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 : (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 h thng ca my lm lnh. 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 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.

IV. Chun b mu DNA Mu DNA ca i tng cn nghin cu c ly trch v tinh sch, sau tr mu 200C.

V. Thit k mi - Mi l nhng on oligonucleotide (18-24 base) b sung vi trnh t trn DNA, gm c mi xui v mi ngc
Mi xui (sense primer hoc Forward) bt cp vi mch khun ca DNA v s ko di

theo chiu phin m.

Mi ngc (anti sense primer hoc Reverse) bt cp vi mch m ca DNA v s ko

di ngc theo chiu phin m.

Trnh t ca mi c thit k sao cho khng c s bt cp gia mi xui v mi ngc,

k c cu trc kp tc + Nhit gn mi ca mi xui v mi ngc khng c chnh lch qu xa (Khng nn

qu nhiu G hoc C ni tip nhau, thng t l G, C nm trong khong 30%< G+C<70% + + Mi phi bo m di bt cp chnh xc. Cc mi c chn phi c trng cho trnh t DNA cn khuych i khng trng vi cc

trnh t lp li trn DNA, mt mi ch bm vo mt v tr nht nh trn gen. + Trnh t nm gia mi xui v mi ngc khng qu ln (<3000 nu

tt nht l di 1000 nu) HNH: S hnh thnh nt ci tc do mi cha trnh t i xng bc hai ch tnh nhit bt mi: - i vi mi 20 nucleotide th nhit gn mi c tnh nh sau: Tm = [2(A+T) + 4 (G+C)] * V d, on mi CAGCAAATATCTGTCCTTAC th nhit gn mi: Tm = [2(6+6) + 4(2+6)] = 560C i vi mi >20 nucleotide th nhit gn mi c tnh theo cng thc: Tm = 22 + 1.46[ 2(G + C) + (A + T)]0C

VI. iu kin phn ng PCR th nghim PCR c th chy c, cn phi c y cc thnh phn sau y: 1. Nc Nc s dng cho phn ng PCR phi tht tinh khit, khng cha ion no, khng cha DNAase, RNAase, enzim ct gii hnNi cch khc l khng cha bt k mt thnh phn no khc.

2. Dung dch m cho phn ng PCR: Dung dch m cho PCR l mt trong nhng yu t quan trng nh hng n cht lng phn ng PCR. Thnh phn dung dch m ca phn ng PCR thng ph thuc vo loi enzim DNA polymerase s dng trong PCR, thng cha mui m Tris HCl 10 mM, KCl50mM v MgCl2 1.5mM. 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, phn ng r nt, ngi ta phi ti u ha phn ng bng cch thm d mt nng MgCl2 thch hp nht. Nng MgCl2 c th dao ng t 0.5-5 mM. Chnh ion Mg2+ gn lin kt dNTP vi DNA polymerase, tng kh nng bm ni ca mi. Nng MgCl2 cao s to nhiu sn phm khng c hiu, nng MgCl2 qu thp s khng to sn phm PCR.

3. dNTP (deoxy nucleoside triphosphate), tc l n v c th tng hp c cc bn sao ca DNA ch. dNTP 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 DNA 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). Mi (primer) Mi l nhng on DNA si n ngn v cn thit cho vic xc tin phn ng dy chuyn tng hp DNA. Chng nhn ra phn DNA cn c nhn ln, bt cp b sung vi mt u ca DNA mu v to ra v tr bt u ti bn. Cc mi ny c chiu ngc nhau, bao gm mt mi xui (forward primer) v mt mi ngc (reverse primer).

Mi l yu t quan trng nht ca phn ng PCR, quyt nh tnh c hiu ca phn ng 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

5. Enzim DNA polymerase (Taq) L enzim xc tc cho qu trnh lp rp cc nucleotide A, T, G, C vo mch DNA mi tng hp, phi l men polymerase chu c nhit cao. Vo thp nin 1960, nh Vi sinh vt hc Thomas Brock n Cng vin Quc gia Yellowstone (Bang Wyoming, M) nghin cu cc vi sinh vt a nhit sng trong sui nc nng 80-900C. ng pht hin mt loi vi khun pht trin mnh nhit cao, c tn l Thermus aquaticus. Hai mi nm sau, cc nh khoa hc ca tp on Cetus (Tp on Cng ngh Sinh hc California) nhn thy rng DNA polymerase t Thermus aquaticus (Taq-polymerase) c kh nng gii quyt vn ca enzim bin tnh sau mi chu k. DNA polymerase chu nhit s dng cho phn ng PCR ln u tin c bn trn th trng l Taq-polymerase. T n nay, mt s vi sinh vt chu nhit khc c khm ph v ngi ta tch chit thm c cc DNA polymerase chu nhit s dng cho phn ng PCR nh Vent- polymerase (Tli-polymerase), Pfu- polymerase,rTth

6. DNA mu (DNA template) l mu DNA m chng ta s khuch i. C th l DNA kp, n, hoc RNA c tch chit t cc i tng nghin cu. DNA khun c nguyn vn cao trnh ln tp cht.

VII. Chu k nhit

C 3 giai on chnh trong phn ng PCR v chng c lp i lp li nhiu ln (chu k) (thng t 25 n 75 chu k).

* Giai on bin tnh (denaturation): tch chui DNA t si i tch thnh 2 si n. Nhit tng ln 94-96C tch hai si DNA ra. Bc ny gi l bin tnh, nhit tng ln s ph v cu ni hydrogen ca chui DNA. Trc chu k 1, DNA thng c bin tnh n thi gian m chui m bo mu DNA v mi c phn tch hon ton v ch cn dng si n. Thi gian giai on ny khong : 1-2 pht.

* Giai on bt cp (annealing): gn cp mi c trng theo nguyn tc b sung. Sau khi 2 si DNA tch ra, nhit c h xung mi c th gn vo si DNA n. Nhit giai on ny ph thuc vo on mi v thng thp hn nhit bin tnh khong 45-60C. S dng sai nhit trong giai on ny dn n vic on mi khng gn hon ton vo DNA mu, hay gn mt cch ty tin. Thi gian bt cp t 1-2pht.

* Giai on ko di (extension): tng hp chui DNA mi ging chui DNA gc. DNA polymerase gn tip vo si trng. N bt u bm vo v hot ng dc theo si DNA. Nhit ko di ph thuc DNA-polymerase. Thi gian ca bc ny ph thuc vo c DNApolymerase v chiu di mnh DNA cn khuch i. Nh mt quy tc 1000bp/ 1 pht . Cc on DNA mi c hnh thnh li c s lm khun tng hp cho cc chu k tip theo. Nh vy s lng bn sao s tng gp 2 sau mi chu k v sau n chu k, tnh theo l thuyt s lng bn sao l 2n VI. Cc ng dng K thut PCR c ng dng rng ri trong nhiu lnh vc khc nhau, t nghin cu khoa hc n sn xut v i sng x hi. Nhng ng dng chnh ca k thut PCR l: - Xc nh cc on trnh t cn nghin cu - Pht hin t bin - Nghin cu qu trnh tin ho phn t - Phc hi cc gen tn ti hng triu nm

- Chn ging vt nui, cy trng - La chn cc cp cha m thun chng trong thi gian ngn - Xc nh cc loi mi, cc loi c hu bng phng php di truyn phn t Y hc Khoa hc hnh s. - Chun on chnh xc cc bnh nhim trng t vi khun, nm, virus - Chun on sm cc bnh gy ra do ung th, cc bnh di truyn - Xc nh huyt thng, truy tm du vt ti phm - L k thut nn cho cc k thut khc nh: RAPD, SSR

Nguyn Th Thu Lan, Nguyn Hong Chng, H Hunh Thu Dng, Hunh Th L

Duyn, Phan Kim Ngc. 2003. Xc nh gii tnh heo bng k thut PCR. Tp ch di truyn & ng dng ,S 4. + Tang v ctv (2000) dng k thut PCR competitive nh lng WSSV (White Spot Syndrome Virus) nhim trong Penaeus monodon Fabricius phn loi giai on nhim WSSV thnh mc nh, trung bnh v nng, t c cch x l thch hp lm gim thit hi trong ngh nui tm (Tang, K. F. J., Lightner D. V.,2000. Quantification of white spot syndrome virus DNA throught a competitive polymerase chain reaction. Aquaculture, 189:1121) + Pht hin nhanh Salmonella spp. trong thc phm (Phm Minh Thu, Phan Thu Dng, Trng Xun Lin v cng s. Vin Pasteur TP.HCM) + p dng k thut PCR sn xut B KIT chun on bnh m trng trn tm, bnh vng

l gn xanh. Vin NC&PT Cng ngh Sinh hc- i hc Cn Th. CC K THUT IN DU DNA (DNA FINGERPRINTING) I. K thut RAPD (Random Amplified Polymorphic DNA) 1. Gii thiu L phng php phn tch a dng DNA khuych i ngu nhin, k thut ny c pht hin vo nm 1990 (Welsh v McClelland; William v ctv.). Dng nghin cu s khc bit di truyn ca nhng loi khc nhau. Mi c s dng trong k thut RAPD l nhng si oligonucleotide gm 10-mer c trnh t sp xp ngu nhin. Cc sn phm RAPD c phn

tch bng cch in di trn gel agarose. H gen ca hai loi khc nhau s cho nhng on bng trn gel khc nhau, t c th so snh s a dng sinh hc ca cc ging cy. 2. Nguyn tc K thut RAPD da trn k thut PCR, bng cch s dng nhng primer ngn (khong 10 nucleotide) c trnh t bit trc, bt cp v nhn bn ngu nhin nhng on DNA c trnh t b sung vi trnh t ca cc primer. Cc on mi oligonucleotide nu bt cp ngu nhin vi c hai mch i din ca mch khun DNA trong khong cch c th khuch i c (di 3000bp) s cho ra nhng on DNA c kch thc khc nhau sau khi khuch i. S c mt ca cc sn phm DNA khc nhau chng t c mt s tng ng hon ton hay mt phn gia DNA b gen v mi. Cc mi dng trong RAPD thng ngn v vy d dng tm c cc on tng ng trn mch n DNA b gen. Do tnh a dng thu c nh RAPD l ng tin cy, v khi c s thay i mt base nit no th n s ngn cn vic tip hp gia mi v DNA mch khun. S mt on nhim sc th hoc s thm bt im gn mi cng nh s xen vo ca mt gen no s lm thay i kch thc on DNA c khuch Dng nhiu primer khc nhau s khuch i nhiu on gen vi kch thc khc nhau, kt qu ny c th hin trn gel vi nhiu bng (band) nhng v tr khc nhau, cc bng a hnh c ghi nhn v t c th v c bn trn mt qun th ang phn ly. Sinh vt cng loi s c kiu gen hon ton ging nhau, s khuych i cc on DNA c kch thc ging nhau khi s dng bt k mi ngu nhin no chy PCR. Sinh vt khc nhau t nhiu s khc nhau v kiu gen _ a dng v sinh hc, th vi mt s mi ngu nhin no cc on DNA c khuych i s c kch thc khc nhau.
Mi

L on ngn oligonucleotide n (10 nu) c tng hp mt cch ngu nhin, c hm lng G+C t 60-70%, v khng c u t b sung. tm s khc bit gia cc loi, ngi ta thng s dng mt s lng ln cc mi ngu nhin. Trn th trng hin nay c rt nhiu mi ngu nhin c tng hp nhn to.

3. Qui trnh thc hin k thut RAPD - Thit k mi - Chun b mu DNA: mu DNA c ly trch phi thun, t tp cht.

- Pha mix cho phn ng PCR vi cc thnh phn theo th t sau: H2O tit trng, Buffer, dNTP tng s, mi xui, mi ngc, Taq polymerase, DNA mu. - Cho vo my lp chng trnh chy ph hp - Kim tra sn phm PCR trn gel agarose vi s c mt ca thang chun - Phn tch kt qu bng phn mm BioPRO. - Xc nh tnh di truyn bng cc phn mm thng dng. Cc s liu thu c cho thy s gn gi hay cch bit di truyn ca cc mu nghin cu. - H s ng dng di truyn c th c tnh theo cng thc ca M. Nei & Li (1979) Sij= 2Nij/ Ni + Nj Ni: s vch ca ging i Nj: s vch ca ging j Nij: s vch chung ca 2 ging i v j 4.ng dng K thut RAPD c ng dng nghin cu a dng di truyn ca cc ging. Pht hin kh nng di truyn lin quan n 1 tnh trng: trong chng trnh ci thin ging, nh chn ging thng quan tm n nhng tnh trng m n biu hin bn ngoi chnh l kiu hnh (phynotype). cc tnh trng c th l tnh khng su bnh, tnh chng chu phn mn Nhng nu vic phn tch di truyn ch da trn kiu hnh thng kt qu d b sai lch, do kiu hnh l tng tc gia kiu gen v mi trng. Chnh v th, nh chn ging cn phi bit tnh trng lin kt vi kiu gen nh th no, phng php RAPD cng gip pht hin c iu ny (Vng nh Tr, 1998) Thit lp bn di truyn: t qun th cha m F1 v qun th phn ly F2, ngi ta c th nh gi cc bng hin din , sau dng phn mm ph bin hin nay l Mapmarker thit lp bn di truyn. S dng k thut RAPD Nghin cu a dng sinh hc ca ging cy c mi huyn G Quao, tnh Kin Giang. Nhm nghin cu s dng 4 mi A02, A04, A11 v A13 trong phn tch a dng di truyn ; kt qu c 49 du phn t c ghi nhn. T v c gin ph h ca cc ging cy ny. Nguyn Hu Hip, Trn Nhn Dng, ng Thanh Sn v Nguyn Vn c. Tp ch Khoa hc 2004:1, trang 105-114. Phng php RAPD c nhng nhc im:

S xut hin cc bng tnh tri, iu khng phn bit c c th ng hp t v c th d hp t. Tnh lp li khng cao do primer l primer ngu nhin v ph thuc iu kin phn ng PCR. Tr ngi trong vic gii thch cc bng c cng tc di chuyn. Nhng u im ni bt ca k thut RAPD: Phng php pht hin nhanh tnh a dng di truyn n gin, khng di hi k thut cao. Tng i r tin so vi cc phng php khc nh RFLP, SSR Khng dng ng v phng x. k thut AFLP I. Gii thiu K thut AFLP (Amplified Fragments Length Polymorphism) c hiu l s a dng ca cc on DNA c nhn ln c nh hng sau khi b ct bi 2 enzim gii hn, s dng nhng phn on DNA lm khun cho phn ng khuch i PCR. AFLP l mt trong nhng k thut in du DNA c pht trin bi Vos v cng s nm 1995. K thut ny l mt cng c hu ch xc nhn nhiu loci ca a hnh DNA m khng cn phi bit trc thng tin v trnh t DNA ca chng (Michelmore v ctv., 1998), phng php ny c th a ra nhanh chng mt c lng a dng di truyn trong v gia nhng qun th vi nhau (Breyen v ctv., 1997; Cervera v ctv.,1996)

II. Nguyn tc Nguyn tc ca phng php AFLP cng ging nh RFLP, im khc bit c bn l AFLP khng cn tin hnh lai phn t (lai Southern blot ), do vy thc hin nhanh hn. K thut AFLP gm hai ni dung c bn l: Ct DNA bng enzim gii hn c b sung cc adaptor c hiu to nn cc on mt ging nhau, c trng cho cc mi chn trc. Cp enzyme thng c dng nht l EcoRI MseI. on adaptor gm 2 phn: phn trnh t li v phn trnh t c hiu cho v tr ct enzyme.

 Khuch i on DNA bng k thut PCR ngt qung vi hai loi mi khc nhau. Mi ca phn ng PCR c thit k da trn trnh t adaptor v cha mt trnh t chn lc khong vi nucleotide. Ch nhng phn on DNA no cha c trnh t adaptor v trnh t nucleotide chn lc mi c khuch i, chnh trnh t chn lc s lm gim s xut hin sn phm PCR v lm n gin qu trnh phn tch. I. K thut AFLP 1. Qui trnh Qui trnh thc hin AFLP gm bn bc:

Bc 1: Digestion Dng 2 enzim gii hn EcoRI (t c = 4096 bases) c trnh t nhn bit 6 base v MseI (thng c = 256 bases) c trnh t nhn bit 4 base, ct DNA khun thnh nhng phn on. Hai enzym gii hn ny c dng sp xp li DNA nh kt hp vi 2 adaptor c cu trc lm cho dy DNA khng tr li hnh dng ban u V tr ct ca enzim

EcoRI = 5-G AATT C-3 3-C TTAA G-5

MseI = 5-T TA A-3 3-A AT T-5

Bc 2 : Ligation Ni vi EcoRI adaptor v MseI adaptor, l nhng oligonucleotide si i gn vo 2 u phn on DNA. Cu trc ca 2 adaptor nh sau : EcoRI-adaptor 5-CTCGTAGACTGCGTACC CTGACGCATGGTTAA-5 MseI-adaptor 5-GACGATGAGTCCTGAG TACTCAGGACTCAT-5 Bc 3 : Tin khuch i - Chy PCR vi cc mi (primer) chn lc, cc mi ny c cu to gm 3 phn : trnh t nng ct (CORE) + trnh t theo enzim (ENZ) + trnh t chn lc (EXT) (theo nh hng ca ngi nghin cu k hiu l NNN, c th l TGA hoc AAC hoc CTG) Nh vy, ch nhng phn on c nu b sung c vi nu chn lc c nhn ln. S khuch i chn lc nh vy lm gim s lng phn on hng ngn ln. Ch nhng on DNA ngn c mt u l primer MseI v mt u l primer EcoRI l c khuch i ng k, cc on c hai u l EcoRI b mt i do s lng t v hai u l MseI t to thnh vng nh DNA do cnh tranh vi primer. Sn phm tin phn ng c pha long v c dng lm vt liu khuch i vi on mi chn lc mu hunh quang.

Bc 4 : Khuch i Sn phm PCR ca bc tin khuch i c dng lm khun cho bc khuch i sau , s dng primer c gn 3 nu chn lc u 3 v ch EcoRI primer c nh du hunh quang u 5. V vy, ch nhng si cha EcoRI c pht hin trn my ABI 310. PCR c xy ra vi thng s miu t bi Cervera v ctv (1996). Phn ng bt u vi nhit cao gn mi ti ho cho mi chn lc. Nhit ny gim dn s lm tng s kt hp. S dng primer gn vi 3 nu chn lc s gip hn ch vic bt cp sai (mismatch), ch nhn ln mt s lng ln nhng bng chuyn bit. Ngoi ra, vic khuch i qua 2 bc c nhng thun li hn khuch i trc tip nh: - Ph din r hn v khng c qu nhiu loi marker.

- Qua bc tin khuch i, to lng ln DNA khun cho s khuch i d dng v hiu qu hn. - Sau khi xong phn ng, mu c bin tnh bng cch thm mt lng tng ng th tch 15l dung dch m Formamide v un nng 5 95oC. 1ml mi mu c np t ng vo ng mao qun (capilary) polymer ABI 310.

Hnh : cc bc thc hin k thut AFLP

2. u v nhc im ca k thut AFLP u im ca k thut AFLP - AFLP khng phc tp nh RFLP nhng vn kho st c ton b gen. - C tnh lp li cao hn RAPD v ch cn s dng mt lng nh DNA ban u (2.5pg-25ng) - Khuch i c chn lc mt lng ln cc on DNA a hnh (polymorphism) trong mt phn ng PCR, ph hp cho vic phn tch a dng gia cc qun th c quan h gn nhau. - Khng cn bit trc trnh t DNA ca gen cn nghin cu, khng cn s dng nhiu loi primer. - L k thut in du DNA cn kh mi l nhng rt hiu qu, c th in du DNA ca bt k ngun gc phc tp no t sinh vt s hch, thc vt, ng vt n con ngi. Nhc im - Tuy nhin AFLP l mt marker tri, iu ny lm hn ch phn bit c th ng hp v d hp. - Qui trnh di, phc tp, tn nhiu thi gian thc hin.

- L thuc nhiu vo thao tc nhng bc u c c ph din cc phn on DNA l tng.

III. ng dng - In du DNA, lp bn DNA marker c hiu qu nht so vi nhng marker khc (Vos v ctv., 1995). - To nhm lin kt di truyn gia cc ging. - nh gi mc lin h di truyn hoc s khc nhau gia cc ging. - L cng c hiu qu pht hin tnh cht a hnh nn d dng nhn bit s khc bit gia cc c th.

Travis v cng s ng dng k thut AFLP nh ga s a dng di truyn gia tt

c nhng qun th ca Astragalus cremnophylax . Chn loi mi c s dng cho ra 325 vch ca 143 c th c thu mu t ba qun th. T kt qu phn tch c, Travis v cng tc vin (1996) c th biu th c im ca s a dng di truyn v cu trc qun th ca chng v a ra hng qun l vic bo tn loi cy ny. (Travis, S. E., J. Maschinski and P. Keim, 1996. An analysis of genetic variation in Astragalus cremnophylax a critically endangered plant, using AFLP marker. Molecular Ecology 5:735-745)
Van Droogenbroek B. et al., 2002. k thut ny c ng dng nghin cu mi

quan h di truyn gia cc ging u trng v u hoang di (Caricaceae) Ecuador (Van Droogenbroek B., Breyne P., Goetghebeur P., Romeijin Peter E., Kyndt T., Gheysen G., 2002. AFLP analysis of genetic relationships among papaya and its wild relative (Caricaceae) from Ecuador. Theor Appl Genet 105: 289-297)
Nguyn Th Loan Anh v cng s s dng k thut AFLP nghin cu a dng

sinh hc cc ging bi (Citrus maxima (Burm.) Merr.) Vit Nam. Kt qu tng s c 60 du phn t ghi nhn c, trong c 6 du phn t xut hin tt c cc ging bi nghin cu, 2 du phn t xut hin vi tn sut 1/40 ging. (Nguyn Th Loan Anh, Tn Khang, Trn Nhn Dng, H Thanh Ton, Tina Kyndt, Godelieve Gheysen, Marcelle Holsters, 2008. Nghin cu a dng sinh hc cc ging bi

(Citrus maxima (Burm.) Merr.) Vit Nam. Hi ngh khoa hc cy n tri quan trng ng Bng Sng Cu Long. NXB Nng Nghip TPHCM).