CC K THUT PCR V NG DNGK 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 chuyenAcid 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