TRNG I HC CN TH

VIN NC V PT CNG NGH SINH HC

--- ---

BI BO CO
MN: DI TRUYN PHN T

K THUT AFLP
(Amplified Fragments Length Polymorphism)

Ngi hng dn Gs.Ts. NGUYN TH LANG Vin la BSCL

Nhm thc hin: L Th Thi Bch (051004) Trn Th M Nhung (051019) Nguyn Th T Quyn (051022) Trn Ch Cng (051005) Bi Minh Trng (051029)

Thng 10/2010

PHN M U

Cng tc lai to ging theo phng php truyn thng c h s nhn chm v c nhiu tr ngi v c ch sinh hc sinh sn ca ging cy trng v vt nui rt phc tp. Kim sot mt s tnh trng ca cc ging ny lm tiu tn nhiu thi gian, chi ph cho chng trnh lai to ging. Hn na, nh u t quan tm nhiu n cc tnh trng lin quan n s lng hn l cht lng. Gn y, nh s pht trin ca sinh hc phn t cng vi s h tr ca marker chn lc cho php pht trin nhng qui trnh da trn kiu gen (khng chu nh hng khi iu kin mi trng bin i) hn l chn lc da trn kiu hnh nh php chn lc truyn thng vn thc hin. Bng k thut AFLP (amplied fragment length polymorphism), mt phng php c hiu qu nhn dng v gii trnh t nhng marker lin kt ny xc nh c im ca chng. AFLP nhanh, n gin khng phc tp nh RFLP nhng vn kho st c ton b gen. K thut ny i hi t lng DNA ban u, khng cn bit trc trnh t ch v lp li phn ng cao, cc mi s dng khng cn c hiu loi (cc mi thng mi c th dng cho hu ht cc loi).

PHN NI DUNG
A. GII THIU
I. K THUT AFLP 1. Khi qut V cn bn, bt c mt chui m DNA no c dng phn bit gia hai c th, hai dng hoc hai ging khc nhau, u c xem nh l mt DNA marker. DNA marker c chia lm bn nhm nh sau:

- PCR based ( ALP, AFLP, SSR, SSCP, STS).

- DNA- khng phi l PCR base nh: DNA/DNA hybrybization: RFLP minysatellite. RFLP thng c h di truyn l Dominant. - Genomic base: Th d SNP dng tch cc vng t bin. - Protein base v proteomic: cc marker chia ra 3 kiu. + Isozyme l enzyme marker + Protein d tr + PTLs protein quantitative luci: protein th hin nh l marker v nh l cc tnh trng trong bn . Ty theo tnh cht ca genomic m chng ta c th p dng cc marker khai thc da dng ha ngun gen pht hin gen, xy dng bn gen. Trong cc marker trn k thut AFLP thuc nhm PCR based c hiu l s a dng ca cc on DNA c nhn ln c nh hng sau khi b ct bi 2 enzyme 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 (DNA fingerprinting) c pht trin bi Vos v cng s nm 1995. Fingerprinting v ang c pht trin kh thnh cng vi hai hng chin lc nghin cu nh sau :

- Fingerprinting trn c s lai, c tnh cht kinh in: Bao gm k thut ct DNA, k thut in di, RFLP c pht hin bi k thut lai vi probe thng qua xt nghim Southern. - Fingerprinting trn c s PCR: Bao gm k thut khuch i DNA in vitro bng primer c bit hay primer ngu nhin. K thut AFLP l mt cng c hu ch xc nhn nhiu loi 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). Nghin cu a hnh ca DNA nh AFLP l m hnh mu trong nghin cu di truyn Menden, do n c th c dng trong vic xy dng bn gen ca cc sinh vt, trong c con ngi chn on v cha tr cc bnh di truyn, cng nh to ra ngn hng gen phc v li ch cho con ngi. 2. Nguyn tc Nguyn tc ca phng php AFLP l da trn c hiu ca cc enzym ct gii hn (restriction enzym RE) i vi v tr nhn bit ca chng trn DNA b gen. S khc bit v tr gia hai c th s to ra nhng phn on ct khc nhau. K thut AFLP gm hai ni dung c bn l: - Ct DNA bng enzyme 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. Khi s dng chng chung vi nhau, nhng enzym ny s to ra nhng on DNA rt ngn, chng c khuch i kh tt, v t c kch thc ti ho (<1Kb) chy in di trn gel polyacrylamide. Do vic thit k primer v k thut

khuch i, ngi ta thng dng phi hp EcoRI - MseI hn l phi hp EcoRI - EcoRI hoc MseI - MseI. - 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. Phng php ny to ra mt lng ln cc band trong in di, lm cho vic pht hin th a hnh thun li hn rt nhiu. S on phn t DNA khuch i c th c kim sot bng cch chn lc base khc nhau v thnh phn nucleotide trong adaptor. 3. u im v nhc im * u im - Xy dng bn di truyn c mt cao ca genome. - 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. II. ENZYME CT GII HN (RE) 1. Khi qut: Trong phn tch genome, enzyme c nhim v cc k quan trng l nhm restriction endonuclease (enzyme ni sinh, phn ct gii hn). l nhm ca Dnase, ghi nhn cc chui trnh t c bit ca cc nucleotide v ct DNA ti v tr c bit. Ngi ta xem n nh cha kha nghin cu k thut recombinant DNA. Enzyme 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. Enzyme 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 enzyme gip vi khun c kh nng ny, chnh enzyme ny chu trch nhim gn nhm metyl (CH 3) 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.

Enzyme ngoi chc nng phn ct, n cn c chc nng kt ni cc phn t DNA li vi nhau. 2. Tn gi ca enzyme 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 enzyme 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. Enzyme 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. Adapter Pst I adapter sequence 1 (96) Pst I adapter sequence 2(97) MseI adapter sequence 1 (A 18) MseI adapter sequence 1 (A 19) Oligonucleoside sequence 5 CTCGTAGACTGCGTACACATGCA-3

3 CATCTGACGCATGT -5 5 GACGATGAGTCCTGAG 3 3 TACTCAGGAGCTGAG- 5

Primer / trc khi khuch i: Pst I primer (96) MseI primer (H 18) Primer/ khuch i chn lc: Pst I primer (90) Pst I primer (91) Pst I primer (98) Pst I primer (99) MseI primer (F 10) MseI primer (F 38) MseI primer (F 39) MseI primer (F 41) MseI primer (G08) MseI primer (G 23) MseI primer (G 24) MseI primer (G 25) MseI primer (G26) MseI primer (G 27) 5 GACTGCGTACATGCACCA -3 5GACGTCGTACATGCAGTT -3 5GACGTCGTACATGCAGAC -3 5GACGTCGTACATGCATGG -3 5GATGAGTCCTGAGTAACAC-3 5GATGAGTCCTGAGTAAACC-3 5GATGAGTCCTGAGTAACCA-3 5GATGAGTCCTGAGTAACAA-3 5GATGAGTCCTGAGTAAACG-3 5GATGAGTCCTGAGTAACAG-3 5GATGAGTCCTGAGTAACAT-3 5GATGAGTCCTGAGTAACGA-3 5GATGAGTCCTGAGTAACGT-3 5GATGAGTCCTGAGTAACCT-3 5 CTCGTAGACTGCGTACATGCA- 3 5 GACGATGAGTCCTGAG- 3

Chui m ca adapter v primer dng trong phn tch AFLP III. K THUT PCR (Polymerase Chain Reaction) 1. Khi qut 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 v ctv., 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. 2. Nguyn l 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 enzyme 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 enzyme 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. 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 enzyme helicase. + Kt hp vi enzyme 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.

3. Cc giai on phn ng PCR 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 DNA-polymerase 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 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.

B. PHNG PHP K THUT AFLP K thut AFLP gm cc bc sau: -Phn lp DNA -Ct bng enzyme ct gii hn v ni bng adapter -Tin khuch i -Khuch i -Chy in di v phn tch kt qu

S chung ca k thut AFLP I. PHN LP DNA 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. Trc ht chng ta phi chun b cht thm d ca marker v b lc DNA lai. l giai on c tnh quyt nh trong phn tch AFLP. V d chng ta cn c DNA t m la vi s lng nhiu v cht lng tt. Trong qu trnh phn lp DNA, ngi Ta phi ly m la t trong nh knh hoc trn ng rung, m ny c nghin bng ci trong Nit lng. Cht m khi li trch DNA c cha SDS,

cht ny c th phn gii mng t bo v mng nhn, phng thch DNA vo dung dch, SDS l mt cht ty kh mnh, n c th bin i protein nhit 650C. S bin i ny lm cho cc DNA gn vi protein tch ri vi DNA cn nghin cu. Protein v cht cn b ca t bo c tch khi DNA bng phenol/Chlorophom. DNA pha lng s dc kt ta bng cch tim Isopropanol. Phn lp DNA bng phng php ni trn ch p dng vi DNA c kch thc ln hn 30 kb c mt t tp cht nh polysachcaric. Chng s gy kh khn khi chy in di trn gel, do chng ta phi ht sc thn trng trong phn lp DNA. DNA c phn lp c th c tn tr m 20 0C. Nhng chng ta phi lu rng, mt cht thay i nh no trong qu trnh ng lnh cng c th lm thay i DNA, trnh ng lnh nhiu ln hoc lm tan bng nhiu ln DNA. Trong qu trnh phn lp ngi ta phi s dng nhiu dung mi. C hai dung mi rt quan trng l EDTA v Tris. Tris( trisma, base) c s dng vi kh nng m rt c hiu qu trong dung mi. ph=8 DNA rt n dnh. Trong mi trng axt, purine rt d b phn hy. cu ni phosphodieste d b ph v to ra kt qu purine ha. Do ngi ta phi kim sot pH ca dung mi mc 8.0 khi dng Tris. DNA c th b hng bi cc enzyme lm cho mu DNA b tp. Ch cn cht gy tp th vt cng gy cho DNA b hng, do vy chng ta phi rt cn trng. ngn nga DNA b phn hy bi enzyme, ngi ta phi s dng thm EDTA. EDTA c th kim gi Mg2+ v lm bt hot n trong dung mi. Khng c Mg2+, enzyme khng th hot ng. II.CT BNG ENZYME V NI DNA BNG ADAPTER * 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.

Digestion:

Eco RI

Mse I

GAATT C CTTAA G
Eco RI

T TAA AAT T
Mse I
7

Dng 2 enzyme 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

AATTC G

AAT

Ligation:
Ni vi EcoRI adaptor v MseI adaptor, l nhng oligonucleotide si i gn vo 2 u phn on DNA. Cu trc ca hai adapter nh sau :
EcoRI Adapter

G CTTAA

TAA T

MseI Adapter

EcoRI Adapter

G AATTC

CTTAA G

TTAA AAT T

MseI Adapter

III. 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 enzyme (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. - Phn ng tin khuch i DNA c thc hin trong th tch 50 l gm: nc ct 2 ln, PCR buffer, MgCl2, mi EcoRI-O v MseI-G, dNTPs, taq polymerase v 5l, DNA qua ct ni.

28 chu k Nhit 94o 94o 72o 1:00 60o 1:00 4o

Thi gian 2:00 0:30

Chu k nhit ca phn ng tin khuch i

IV. 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. Thnh phn phn ng gm: nc ct 2 ln, PCR buffer, MgCl2, mi EcoRIATC c nhum vi mu NED hoc HEX v MseI-GGC, dNTPs, taq polymerase v 5l DNA qua tin khuch i. nh du phng x. Sau thm 15l dung dch m formamide vo 1,5l DNA v un nng 5 pht 950C. 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 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.

Chu k nhit ca phn ng khuch i

V. IN DI V PHN TCH KT QU Trong qu trnh in di do c in tch v khi lng khc nhau, nhng phn t v nhng ht trong mt hn hp s chuyn ng vi tc khc nhau v phn tch thnh nhng tiu phn khc nhau. Nhng on DNA c kch thc khc nhau cng s chuyn ng vi tc khc nhau trong in trng, to nn nhng tiu phn khc nhau. C nhiu phng php khc nhau c th pht hin cc bng DNA sau in di. * Mt s yu t nh hng n qu trnh in di DNA:
- Bn cht v nng gel: Gel Polyacrylamide (PA) c s dng u tin

bi Raymond v W EINTRAU (1959). Gel c tch cht tr v n nh v mt ha hc. Gel c to thnh qua phn ng polymer ha t cc monomer ca acrylamide v N, N-methylen bis acrylamide khi c cht xc tc l ammonium persulfat (APS) v TEMED trong phn ng . Kch thc l ca gel PA c th iu chnh bng cch thay i nng tng s acrylamide v mc lin kt ngang. Gel PA bin tnh c polymer ha vi s c mt ca cc thnh phn nh urea

hay l foocmamide. DNA bin tnh di chuyn qua gel ny vi tc gn nh c lp vi thnh phn v trnh t ca baz. Gel PA bin tnh thng c s dng trong vic tinh khit cc mu d(probe) DNA c gn phng x , phn tch cc sn phm phn ct bi nucleaza S1 v phn tch cc sn phm ca qu trnh gii trnh t DNA. Gel PA khi c pha ch vi mt s ha cht khc to thnh mt cht c tn gi l POP, dng lm mi trng in di trong mao qun cc my gii trnh t bng mao qun. y l nhng my gii trnh t t ng rt hiu qu nhng li qu t. Trong iu kin Vit Nam hin ti th chng ta ch tham kho kh nng phn tch ca gel PA trn bn gel. Phm vi phn tch ca gel PA bin tnh c th hin bng sau: Nng % gel PA/ure 4 6 8 10 12 Kh nng phn tch(bp) >250 60- 250 40- 120 20-60 10-50

Tuy gel acrylamide t v c hn so vi gel agarose nhng n c nhng u im ni bt so v gel agarose: Kh nng phn tch rt tt, c th phn tch c nhng on DNA ch khc nhau 1bp. Chy c vi in p cao nn tit kim c thi gian. - Hiu in th v cng dng in. - Quan h gia hiu in th v chiu di gel: to ra mt in trng cn phi c mt hiu in th. Hiu in th c tnh bng tch ca in trng vi chiu di phn tch. Nh vy, gel cng di th hiu in th s dng cng phi ln to ra c mt in trng nht nh trn ton b bn gel.

- Nhit i vi qu trnh in di: Qu trnh in di s sinh ra nng lng m hu ht nng lng ny chuyn thnh nhit nng. Nhit thch hp nht cho s phn tch cc si DNA trn gel PA bin tnh trong khong 500C. Nhit ny c iu khin bng vic tng gim cc gi tr hiu in th v cng sut khi in di. * Thnh phn dung dch m in di: S di chuyn ca DNA trong in trng b nh hng bi thnh phn v lc ion ca m in di. C mt vi loi m khc nhau dng cho in di DNA nh TAE, TPE, TBE. Nhng m ny c cha EDTA pH=8 vi Tris- acetat (TAE), Tris- borate(TBE) hoc Tris- Photphat (m TPE) c nng xp x 50ml (pH =7.5-7.8). Hin nay th m TBE c s dng rng ri hn c v s n nh v thnh phn ca n. * Phn tch sn phm AFLP: sau PCR, mt th tch formamide dye c thm vo phn ng. Mu c bin tnh bng nhit 950C trong 3 pht v t ngay vo . Sn phm AFLP c in di trn gel polyacrylamide bin tnh (19:1 acrylamide: bisacrylamide; 7,5 M urea; 0,5 X TBE buffer ). Bn gel c c nh trong axit axtic 10%, nhum trong bc nitrat 1%, hin trong natricacbnat 2,5% v c nh li trong axit axtic 10%. C. MT NG DNG CA K THUT AFLP C tra (Pangasius hypophthalmus) l loi c c gi tr kinh t cao v c nui trng ph bin khu vc ng nam . Vit Nam, c tra c nui nhiu cc tnh ng bng sng Cu Long. Thi gian gn y, c c di ging thun ho nui mt s tnh pha Bc. C thng phm c gi tr trn th trng c trong v ngoi nc. Hin c nc c trn 14 doanh nghip xut khu c tra, ba sa vi kim ngch hng nm trn 60 triu USD. Nng cao hiu sut nui trng tng li nhun lun l mc tiu ca cc nh sn xut. Bn cnh nhng nghin cu nhm tng hiu qu sinh sn, dinh dng, vic ci to ging to nhng dng c c

nng sut cao c ch trng. Chng trnh lai to, chn ging truyn thng lun ng vai tr ch o trong ci to ging. Nh vo k thut cho php tuyn chn ng thi trn ton b h gen v to ra mt s lng ln ch th c th chuyn i thnh ch th c hiu v tr n gin (simple locus-specific markers) m khng cn bit trc thng tin ca trnh t c hiu . Pht trin ch th AFLP lin quan tnh trng quan tm c th da trn s phn b ngoi hnh ca hai qun th i lp nhau hoc da trn a hnh di truyn hai nhm c th i lp nhau. Phng php ny c u im khng cn thit lp bn di truyn v to ra cc ph h c bit, nn thch hp cho pht hin v tr tnh trng nh lng (QTL) trc tip t cc qun n thng phm c tui thnh thc di nh c tra (2-3 nm). Vi mc ch pht trin ch th AFLP trc tip t qun n thng phm, phn tch a hnh AFLP gia hai nhm c tra c trng lng cc ln v cc nh.

a hnh AFLP gia nhm c ln v nhm c nh ca t hp mi E-ACA/MCAG. ( 1,2: Bng AFLP xut hin c 2 nhm c vi tn s khc nhau) Tng cng c 204 bng AFLP c to ra t 43 t hp mi, trung bnh l 4,74 bng AFLP/mi. Tn sut xut hin cc bng AFLP hai nhm c tra l khc nhau nn chng ti tnh tn s khc bit gia hai nhm. Kt qu c th hin bng 2.

Bng 2: Tn s khc bit ca cc bng AFLP gia hai nhm c tra Nhng bng AFLP c tn s khc bit trn 50% c th hin bng 3.

Bng 3: Cc bng AFLP c tn s khc bit trn 50 %. Kt qu trn cho thy, ch c 8 bng AFLP c tn s khc bit trn 50%, chim 3,92% tng s bng AFLP. kim tra mc n nh v tin cy ca cc bng AFLP, ngi ta tin hnh phn tch cc bng AFLP ny vi s lng mu ln hn (18 mu). Kt qu cho thy, bng AF 6-1 ca t hp mi E-AGG/M-CTT v bng AF 7-4 ca t hp mi E- ACC/M-CAA t chnh lch gia 2 nhm c th nghim l 56% v 50%. Do c tnh n nh tng i, r nt v u th cho nhm c c trng lng nh, hai bng AFLP ny c tch dng v c trnh t

nhm mc ch xy dng 2 ch th AFLP lin quan ti tnh trng tng trng ca c tra. Phn tch a hnh AFLP c tra bng 54 t hp mi chn lc cho thy t l a hnh trong loi tng i thp, trung bnh 1,35%. Tuy nhin, t l ny tin cy trong vic xc nh cc ch th lin quan tnh trng c ngha kinh t. So snh a hnh AFLP gia 2 nhm c tra c trng lng 2 cc cho thy 8 bng AFLP c tn s khc bit trn 50%. Tuy nhin ch c 2 bng: AF 7-4 ca t hp mi E-ACC/M- CAA v AF 6-1 ca t hp mi E-AGG/M-CTT c tnh n nh, v vn gi c t l ny khi kim tra trn s lng mu ln hn. Hai bng ny s c s dng tuyn chn li trong qun n.
Nguyn Thu Thu, Nguyn Th Diu Thu, Nguyn Vn Cng Vin Cng ngh sinh hc, Vin Khoa hc v Cng ngh Vit Nam

PHN KT LUN

Marker phn t ng vai tr quan trng trong vic xc nh v ci tin di truyn nhiu loi cy trng v vt nui. Nh markers phn t chng ta c th nh gi c s a dng sinh hc, xy dng li ph h v hiu cu trc, s tin ha v mi quan h gia cc c th trong qun th cy trng. H thng marker phn t cho bit s bin thin v trt t ADN trong h genome. Trn th gii, cc nghin cu v di truyn s dng nhiu loi marker khc nhau nh RFLP, RAPD, SSR v ISSR, trong c AFLP. AFLP l s a hnh cc on ct khuch i, l k thut kt hp gia RFLP v PCR. AFLP s dng enzyme ct gii hn ct DNA b gen, dng nhng phn on DNA lm khun cho phn ng khuch i PCR. AFLP c th dng phn bit cc c th rt gn nhau, thm ch ngay c nhng dng ng gen. S khc nhau trong chiu di cc on khuch i c th do nhng thay i ca cc base trong vng trnh t mi, hoc thm, mt on gia hai v tr ct. AFLP nhanh, n gin, khng phc tp nh RFLP nhng vn kho st c ton b gen. K thut ny i hi t lng DNA ban u, khng cn bit trc trnh t ch v lp li phn ng cao, cc mi s dng khng cn c hiu loi (cc mi thng mi c th dng cho hu ht cc loi). Tuy nhin AFLP l mt marker tri, iu ny lm hn ch phn bit c th ng hp v d hp. AFLP c s dng trong vic xc nh tnh a hnh gia cc cc ging loi, 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. ng thi thng tin di truyn t cc vt liu nghin cu s gp phn gip nh hng cho cc php lai trong cng tc chn ging.