Phn loi vi sinh bng Sinh Hc Phn T

Vietsciences- Dng Vn Hp, Nguyn Ln Dng 1. 27/02/2007 Khi qut v cc phng php phn loi vi sinh vt truyn thng:

Trc y cng tc phn loi vi sinh vt vn da cn bn trn cc c tnh hnh thi, sinh l v ha vi sinh vt: nhum, hnh dng t bo khun lc, kh nng di ng, nhu cu dinh dng, kh nng sinh acid trong mi trng cng nh sc t to thnh v.v..Cc c trng ny i khi cng bc l nhng hn ch do cc c tnh c dng cho nhm vi sinh vt ny (Enterobacteriaceae) nhng li khng c ngha i vi nhm khc (vi khun gram m - gram negative bacteria). Hn ch ca cc phng php phn loi truyn thng dn n nhiu trng hp phi xc nh li tn phn loi ca mt s vi sinh vt. T trc n nay, n v c bn ca nh tn vi sinh vt l loi, bao gm nhm cc c th c mc tng ng cao v cc c im hnh thi. Cc phng php da trn cc phn ng sinh ha: T nhng hn ch ca vic xc nh cc c tnh hnh thi dn n nhiu nghin cu tp trung vo cc phn ng sinh ha c trng cho cc vi sinh vt ring bit. S khc bit ca cc phn ng c ngha cho phn loi cc vi sinh vt. - API20E KIT, nguyn tc: da vo 20 phn ng khc nhau. Ni chung hin nay c nhiu ni vn dng k thut ny nhng nhn chung kt qu cng cn nhiu sai s do nhiu nguyn nhn khc nhau: c th trong cc trng hp gene quyt nh phn ng sinh ha nm trong plasmid li b mt do nhiu nguyn nhn khc nhau nh tui t bo, lng ging cy hay thay i trong qu trnh nui cy dn n sai khc v lm sai kt qu. - Phn bit bng thc khun th: Cc vi khun c mn cm vi thc khun th khc nhau. C thc khun th xm nhim lm tan t bo ngay lp tc sau thc khun th nhn ln thnh cc ht trong t bo ch, trong khi vi mt s vi khun th thc khun th xm nhim nhng li khng lm tan t bo vi khun v chng cng tn ti vi t bo vt ch. Da vo s khc bit ny m ngi ta dng cc thc khun th khc nhau phn bit cc i tng vi khun nghin cu. Tuy nhin, phng php ny cng bc l mt s nhc im kh gii quyt l cc c tnh mn cm ca vi khun vi thc khun th li thay i do iu kin ngoi cnh hoc l vi khun li c mc mn cm khc nhau i vi cc thc khun th khc nhau. Mt khc na, thc khun th rt d thay i cc c tnh do cng lm thay i c ch xm nhim vo vi khun ch.

- Phn bit theo Typ huyt thanh: y l phng php c dng kh lu nhng rt hiu qu v hin vn ang c s dng (v d nhm vi khun Bt). Nguyn tc l da vo nhm quyt nh khng nguyn trn t bo vi sinh vt (b mt t bo tin mao hoc protein v). u th ca phng php ny l cc khng huyt thanh c dng bit ha nhiu chi khc nhau, trong nhiu trng hp c trng cho loi. Ni chung y l phng php kh n nh nhng hn ch ch yu ca phng php ny ch: yu cu k thut sn xut khng huyt thanh v tiu chun ha phn ng khng huyt thanh khng ng nht ti cc phng th nghim v tnh n nh gia cc ln lp li. - Phn bit bng loi hot cht khng khun (Bacteriocin): Bacteriocin bn cht l peptid khng khun sinh ra bi vi khun chng li vi khun khc. Nh vy, loi vi khun to ra loi bacteriocin no th c kh nng khng li chnh bacteriocin . Bi vy c nhiu loi vi khun c phn loi da vo kiu bacteriocin. Kt lun: C nhiu phng php truyn thng c s dng trong nhiu nghin cu phn loi nhng khng c phng php no t ra vn nng thch hp cho mi i tng vi sinh vt, tnh chnh xc ch c th t c khi kt hp nhiu phng php khc nhau. 2. Cch tip cn phn loi hc vi k thut sinh hc phn t: Ngy nay nhng tin b trong sinh hc phn t m ra kh nng ng dng hu hiu trong phn loi hc v nghin cu a dng vi sinh vt. Nu nh cc phng php truyn thng ch tp trung trn mt s i tng vi sinh vt th phng php sinh hc phn t c th p dng trn mi i tng vi sinh vt. Ni chung cc phng php sinh hc phn t tp trung vo cc k thut ch yu l: + Phn tch acid nucleic. + Phn tch protein. + Phn tch lipopolysaccharid. + Ha phn loi hc. Trong phn ny chng ti tp trung gii thiu mt s k thut phn loi lin quan n acid nucleic. Cc phn sau chng ti ln lt gii thiu cc phng php lin quan n polysaccharid, lipoprotein v ha phn loi. 2.1. Phn tch acid nucleic: K thut phn tch acid nucleic lin quan ch yu n phn tch kch

thc, cu trc acid nucleic, mi tng quan v trnh t acid nucleic thng qua gii trnh t ADN v lai ADN. a. Phn tch ADN plasmid: Phng php phn tch y bao gm tch plasmid, so snh cc loi plasmid v kch thc sau tinh sch plasmid v x l bng enzym ct hn ch, sau in di trn gel agarose v so snh s a hnh ca cc mnh ct phn bit cc chng vi sinh vt vi nhau.

Plasmid vng

Streptomyces v Borrelia

nhim nhin, khun khun

Plasmid l nhn t di truyn ngoi nhn v sao chp c lp vi sc th. Cu trc plasmid l si i ADN khp kn theo vng. Tuy c nhiu trng hp ngoi l l si kp ADN mch thng (i vi vi Borrelia v Streptomyces). Plasmid c tm thy hu ht cc vi v mt s t cc vi sinh vt nhn thc bc thp nh nm men.

+ Tch plasmid:

Thng thng lng plasmid c trong t bo chim < 5% tng s acid nucleic ca t bo. Nh vy, ta phi thc hin php tch plasmid ra khi ADN ca nhim sc th. Ni chung c nhiu phng php tch plasmid t vi khun nhng nguyn tc chung l ph t bo vi khun dng enzym, siu m hay trong dung dch kim c cht ty ra l SDS hoc TritonX-100, sau l vic loi ADN ca nhim sc th v protein. Thng thng dng phng php kt ta vi axetat. Lc ny phn ln cc thnh phn ADN nhim sc th v protein ca t bo b kt ta b loi bng li tm v plasmid nm trong dch trn ta c tch khi kt ta vi ethanol hay isopropanol. y l phng php c hiu qu tch plasmid, trn thc t hiu qu tch cc plasmid c kch thc nh cao hn nhiu so vi cc plasmid c kch thc ln (>100 Kb). Vi cc plasmid c kch thc ln cn cc thao tc nh nhng trnh t gy do cc nguyn nhn c hc. Vi cch tch plasmid nh trn th sn phm thu c c ln cc on ADN c kch thc khong 500 bp v nhim ARN. trnh nhim ARN th cn x l vi RNase (ribonuclease A). Tuy nhin, c th tch theo cc phng php nh: - Tch bng Caeseium chloride. - Tch bng KIT QIAGENE. - Tch bng k thut khc. + in di plasmid trn gel agarose:

Sau khi thu c plasmid th phi tin hnh kim tra trc khi phn tch kt qu in di trn gel agarose bit ph plasmid v kch thc ca chng. Khi tin hnh in di th nng gel khong 0.7- 1% vi mt trong cc m sau: TAE (40 mM Tris acetat 1mM EDTA, pH 8.0), TBE (89 mM Tris borat 89 mM boric acid, 2 mM EDTA, pH 8.0) v TPE (80 mM Trisphosphats 2 mM EDTA, pH 8.0). Sau khi in di, gel c nhum vi ethidium bromide v pht hin di tia UV (310 nm). Nng ethidium bromide khong 5 mg/l v thi gian nhum l 10 pht. Sau c ra mt ln nhanh bng nc loi ion trc khi c nhn di UV v chp nh lm ti liu. Mt s vn cn lu khi phn tch plasmid: trn gel thng thng plasmid c thy 3 dng: dng siu xon, dng t gy v dng mch thng khi b ct bi endonuclease. Dng di chuyn tc cao hn l siu xon (Hnh 1.1).

Hnh 1.1. Di chuyn ca plasmid mch thng (L) v siu xon (OC) khi in di i khi vic tch plasmid cng phc tp c bit trong nhng trng hp plasmid c kch thc nh c ln cc mnh ADN ca nhim sc th. Trong mi trng hp u c nhim cc RNA v chng di ng nhanh nht tr cc trng hp plasmid nh vy khng c nhm ln gia plasmid siu xon v dng chnh plasmid t gy v plasmid khc. Mt ch khc l trnh nhm ln khi so snh kch thc gia cc plasmid siu xon v dng dui xon hay mch thng. Thc t l ch c th so snh cc plasmid siu son vi nhau v kch thc. Khi tin hnh phn bit gia cc chng vi khun th ng nhin l ch c th thc hin so snh gia cc chng cng c plasmid vi nhau. Cng nn ch l khng phi cc chng u ging nhau v c tnh min dch nu c chung kt qu phn tch plasmid nh nhau. Php phn tch s c hiu qu trong cc mu c nhiu loi plasmid, tuy nhin cng phi tnh n vic cc plasmid cng c th b mt trong qu trnh bo qun. + K thut du vn tay (finger printing) phn tch plasmid vi enzym ct hn ch: Cho n nay, ngi ta tm thy hn 400 enzym ct hn ch. Mc ch ca k thut ny b sung cho k thut so snh ph kch thc plasmid trn. Tc l ngi ta c th phn bit c s khc nhau ca cc plasmid c cng kch thc. Nh vy, khi x l plasmid vi mt hay kt hp mt s enzym ct hn ch th kt qu s thu c l cc on ADN c ct c kch thc khc nhau. Kt qu ny c lp li cao, do d s dng khi so snh cc mu khc nhau. Hin nay, c nhiu enzym ct hn ch c sn xut v tiu th trn

th trng, cc enzym ny c im nhn bit thay i t 4-6 cp nucleotid. Thng thng x l enzym trong 1 gi sau mu c pht hin trn gel agarose hoc polyacrylamid Tu thuc vo kch thc ca cc mnh ct m s dng agarose c nng khc nhau: Bng 1.1. Nng agarose v kch thc mu ADN tng ng. Nng agarose (%) 0.3 0.5 0.8 1.0 1.2 1.5 2.0 Kch thc ADN (kb) 1-7 0.7-45 0.4-20 0.3-10 0.2-8 0.2-6 0.1-5

Theo kinh nghim ca nhiu tc gi (Grimont-1991) ph cc bng ADN c ngha cho so snh khng nn di 10 bng v nn c c bng kch thc nh v kch thc ln. Tuy nhin, cc bng ln khng nn vt qu 10 do bng c kch thc ln nh vy kh di chuyn trn gel. Trong cc trng hp so snh th nn dng thang chun ADN so snh kch thc v php phn tch du vn tay cho cc ln phn tch khc nhau. Nh trnh by trn, nu nh phn tch cc chng vi sinh vt da vo kch thc plasmid c ngha i vi cc i tng c nhiu plasmid th phng php x l enzym ct hn ch li c ngha ln trong cc trng hp nghin cu dch t hc trn cc chng c cng ph plasmid hay plasmid c kch thc ging nhau. Ngi ta ng dng k thut ny trong nghin cu chng E.coli (0157: H7) gy bnh t thc phm (Hamburger). Cc plasmid t cc chng khc nhau th c ph khc nhau v cc c im ct bi enzym ct hn ch. Kt qu to ra cc mnh ADN c trng cho c th lm c s cho php so snh. Tuy nhin kh c th so snh kt qu thu c t cc phng th nghim khc nhau, s d nh vy l do khng dng cng mt loi enzym ct hn ch. Mt l do na cng cn c cp n l s xut hin cc t bin ngu nhin ti v tr enzym ct, kt qu ny to ra s khc bit v ph thu c t cc mnh ct. b. Phn tch ADN nhim sc th: Phng php phn tch y bao gm vic tch ADN ca nhim sc th v ct bng enzym ct hn ch phn bit gia cc vi sinh vt vi nhau. Mt ch khi tch ADN nhim sc th phi thao tc nh nhng trnh t gy do nguyn nhn c hc. Ni chung cc mnh ct nn c kch thc nh hn hoc bng 50 kb. Vic tch cc mnh ct c thc hin da vo k thut in di trong trng xung in (PFGE = Pulsed Field Gel Electrophoresis ).style="text-align: justify; margin-top: 6.0pt"> Nh

vy k thut du vn tay khng ch p dng trong trng hp trn t bo mang plasmid m k thut ny cn c s dng vi nhim sc th cho mi trng hp.rng hp.ng hp.ng hp. Lin quan n vn ny phi k n k thut BRENDA: phn tch kt qu x l enzym ct hn ch vi vi sinh vt. y cch chn loi enzym ct hn ch v cng quan trng, theo cch chn ny c th to ra qu nhiu mnh ct nn khng th phn bit c cc bng ring r trong khi i vi cc trng hp khc li to ra qu t mnh ct c kch thc ln kh tch theo cc k thut in di hin c. Cc vi sinh vt khc nhau c t l GC khc nhau (dao ng t 25-75%) cc mnh ct thu c sau khi x l enzym ct hn ch rt khc nhau. Theo Nei M. v Li W. H. (1979) th s mnh ct c th thu c tnh trn l thuyt l: a = (g/2)r1 x {1-(g/2)}r2 Trong : g - t l GC ca ADN r1 - s cp GC r2 - s cp AT trong v tr ct ca enzym gii hn s dng a - s v tr ct khi s dng enzym ct hn ch. Mt khc, ngi ta cng cn c vo kt qu nghin cu cc v tr ct ca b gene vi sinh vt lm c s cho cch chn enzym ct. Thng thng cho mi php phn tch ngi ta ghi c s cc bng ct bi enzym v tnh ton kch thc cc mnh to thnh lm c s cho cc php phn tch v sau. Tuy nhin, mt trong nhng nguyn nhn hn ch cho php phn tch trn l cc phng php tch ADN thng thng u dn n thay i kch thc do t gy ADN nhim sc th, mt khc s c mt ca plasmid ln cng to ra s khc bit gi trong kt qu phn tch, khc phc nhc im trn ngi ta phi thc hin php phn tch ADN nhim sc th nguyn vn pht hin cc ngun ADN tp nhim ln vo kt qu phn tch. + K thut in di trong trng xung in (in di trong trng in thay i, PFGE) - Nguyn tc: Trc tin cc mu a vo phn tch phi c x l trc bng loi enzym ct hn ch m c rt t im ct. Kt qu phi to ra c cc mnh c kch thc ln (50 kb 12 Mb), vi kch thc ny khng th in di theo phng php thng thng m ch c th tch ra bng k thut PFGE (t PFGE (t PFGE (ut PFGE (Pulsed-Field Gel Electrophoresis ). K thut PFGE ln u tin c Schwartz va Cantor (1984) gii thiu. Tuy nhin sau c nhiu tc gi m t li k thut ny, tuy c sai khc t nhiu nhng c s l thuyt ca cc phng php ny l nh nhau: Mc ch ca k thut ny l tng kh nng di ng ca

cc mnh ADN c kch thc ln trong in trng, do cc thnh phn gel v m hu nh khng thay i theo cc phng php in di thng thng. Tuy nhin theo phng php thng thng th s in di lin quan n s di ng ca cc mnh ADN c kch thc khc nhau trn gel agarose trong mt trng in khng i. Kt qu y l phn t ln kh di ng hn phn t nh qua mt xch agarose, to ra s tch bit trong trng in di. Trong trng hp PFGE lc lm thay i kh nng di ng li l trng in tch thay i lin tc dn n cc mnh ADN c kch thc khc nhau thay i hng di ng. Nh vy kt qu l cc mnh c kch thc khc nhau s c kh nng di ng khc nhau. Kch thc cng ln th mc di ng cng chm. S di ng khc nhau ca ADN ch yu ph thuc vo kch thc ch khng phi do gel agarose nh phng php in di thng thng, hnh 1.2.

Hnh 1.2. S dng k thut PFGE phn tch nhim sc th sau khi x l vi Sma I vi 7 chng Haemophilus nluenzae. Tip theo cc m t ca Schwartz va Cantor, Smith v Codemine (1990) cp n s di chuyn ca cc mnh ADN khc nhau l hm s tuyn tnh vi kch thc ca chng. Trn c s c th iu chnh cc xung in v in trng. Tuy nhin cc nhn t khc cng c mi tng tc ln nhau v nh hng n kh nng di ng ca ADN nh: nhit , in th, nng agarose cng nh nng ion trong m. - Chun b ADN cho PFGE: Chun b ADN t nhim sc th khng ging nh cc phng php chun b ADN plasmid. Mt vn thng xy ra l s t gy ngu nhin ADN dn n sai khc trong kt qu phn tch. hn ch iu ny ngi ta phi thc hin k thut tch ADN NST khi t bo mu agarose c nhit nng chy thp LMT (Schwartz va Cantor 1984 v Smith, Klco 1988). Theo phng php ny hn dch t bo (dch nui cy hoc dch huyn ph) c trn vi LMT agarose ti 37oC. Hn

dch u tin c x l vi enzym v cht ty ra tch ADN NST khi thnh, mng t bo, RNA v protein. Sau l x l vi EDTA, bt hot nuclease t bo vi proteinase K v N-lauroylsarcosine loi cc thnh phn khc ca t bo. Kt qu l phn LMT agarose c cha ADN NST c dng lm mu chy gel 0.5-1.0% (nn lm tan 65oC) v a ln mu chy gel PFGE, ti liu ca Smith (1988) m t chi tit cch chun b mu t cc t bo c v khng c thnh t bo: vi khun, nm men, nm si, t bo thc vt v ng vt. Ni chung vi cc trng hp c thnh t bo th cn n enzym ph thnh t bo. Lng ADN NST thng dao ng trong khong 0.5-20 g ADN l thch hp. Nu qu nhiu ADN th khng th phn tch c, cn qu t th cng khng th pht hin c cc bng ADN trong kt qu phn tch. Mu ADN dng cho k thut PFGE c th c gi 1 nm trong lnh c cha 0.5M EDTA. - Sau khi thu c ADN NST trong mu LMT agarose th tin hnh x l vi enzym gii hn loi c t im ct. Tuy nhin mu u tin phi c x l vi PMSF bt hot protein K sau PMSF li phi loi b sau mt s ln ra vi cht ty ra v EDTA. Sau ch cn phn nh mu trong agarose c x l vi m v enzym ct hn ch qua m trong tube v sau ton b mu ny c s dng tch trong trng xung in. Bng 1.2 di y lit k cc enzym ct hn ch c dng cho phn tch PFGE. Bng 1.2. Mt s enzym ct hn ch c dng cho k thut PFGE. Enzym AatII ApaI ClaI MluI NarI NheI NotI NruI PvuI SacII SalI SfiI SmaI XhoI V tr ct GACGT/C GGGCC/C AT/CGAT A/CGCGT GG/CGCC G/CTAGC GC/GGCCGC TCG/CGA CGAT/CG CCGC/GG C/TCGAC GGCC(N)4/NGGCC CCC/GGG C/TCGAG

- ng dng ca k thut phn tch PFGE: K thut PFGE c s dng cho nghin cu trn nhiu i tng khc nhau (xem bng 1.3). Bng 1.3. Minh ho cc chng vi sinh vt c phn tch da trn kt qu PFGE thu c t cc on nhim sc th. Vi sinh vt nghin cu Ti liu tham kho 1. Acinetobacter baumannii Gouby et al (1992) 2. 3. 4. 5. 6. 7. 8. 9. Brucella spp Campylobacter hyointestinalis Campylobacter jejuni CADNida arabicans CADNida prapsilosis Coxiella burnettii Enterobacter cloacae Enterococcus faecium Allardet, Servent et al (19980 Salama et al (1992) Yan et al (1991) Vasquez et al (1991) Carruba et al (1991) Heizen et al (1990) Haertl ADN BADNlow (1993) MirADNa et al (1991) Bohm ADN Karch (1992) Brosch et al (1991) Herrmann et al (1992) Zhang et al (1992) Bygraves ADN Maiden (1992) Boukadida et al (1987) Sodati ADN Piffaretti (1991) Prevost et al (1992) Single ADN Martin (1992)

10. Escherichia coli 11. Listeria monocytogenees 12. Leptospira spp 13. Mycobacterium tuberculosis 14. Neisseria meningitidis 15. Psedomonas aeruginosa 16. Shigella spp 17. Staphylococcus aureus 18. Streptococci ssp

Mi mt i tng c c trng ring v kt qu phn tch PFGE c dng cho nghin cu so snh vi nhau v s tng ng. i nhiu i tng c nhim sc th th khng nht thit phi thc hin vi mi nhim sc th m ch cn trn mt s nhim sc th m thi. V

v vi i d

trong trng hp ca C.albicans Monod (1990) ch cn thc hin php phn tch vi mt trong 4 t hp ca mt s nhim sc th l . Cho d theo cc cch chun b nhim sc th khc nhau th kt qu ca php phn tch PFGE u ging nhau. Php phn tch PFGE c ng dng cho cc nghin cu mc loi vi loi. Mt s im cn lu khi s dng k thut PFGE: + K thut ny i hi phi c trang thit b chuyn dng, k thut cng nh kinh nghim v vic tch nhim sc th i khi gp kh khn trn mt s i tng vi sinh vt. + K thut PFGE khng nhy pht hin nhng sai khc nh gia cc mu, c bit khi thc hin vi mt enzym th kt qu c th ging nhau nhng khng chc chn l hai mu ging nhau hon ton. iu c ngha l k thut ny nn dng xc nh s khc nhau cn vic xc nh s ging nhau th khng tin cy. Tuy nhin ngay c khi xc nh s khc nhau i khi cc mu c plasmid nh hay thc khun th cng cn xt n v chnh ngun ADN ny cng to ra nhng sai khc gi. + S khc bit gia cc mu c th pht hin c khi dng cc enzym ct hn ch khc nhau. Tm tt: Phng php phn tch plasmid l phng php c dng ph bin trong cc phng th nghim chun on vi sinh vt. Vic kt hp gia phn tch plasmid vi s dng enzym ct hn ch s to c s phn tch chnh xc vi cc thng tin ng tin cy i vi cc mu phn tch. Tuy nhin, khi phn tch cc mu da trn plasmid th cn lu l cc plasmid c th b mt qua cc th h phn chia, dn n sai lch cc kt qu nghin cu. Khi thc hin php phn tch PFGE vi cc plasmid ln s khc phc c hn ch ca cc php phn tch hin ti vi plasmid nh nh trn. Phng php PFGE hin ang c dng rng ri ti cc phng th nghim vi sinh vt, p dng trn cc i tng d nui cy c th cho cc kt qu tt hn nhng hn ch v gi thnh thit b cng nh yu cu cao v k thut v kinh nghim. Mt khc khi s dng cc enzym ct hn ch him c th s kh pht hin c mc sai khc. Nh vy s kt hp gia cc php phn tch plasmid, PFGE v lai ADN s cho kt qu tin cy hn. 2.2. Lai ADN Nguyn tc c tin hnh nh sau: ADN tng s c tch ra v x l vi enzym ct hn ch (c trng hp khng cn x l vi enzym ct hn ch) sau in di trn gel agarose v chuyn ln mng lai. Mu d (probe) c chun b v lai vi mng ADN trn. Kt qu php lai cho bit s khc bit gia cc mu ADN khc nhau.

Php lai c tin hnh y da vo cu trc si i ADN t hai si n vi cc cu lin kt hydrogene theo nguyn tc b sung. Bt u l t gy cc lin kt hydrogene do ho cht (kim) hay bin tnh nhit, lc ny hai si n ADN tch nhau c gi l trng thi bin tnh ADN (denature). Nu ti thi im ny ngi ta cho vo mt ADN nh du bin tnh (mu d) sau to iu kin hi bin xut hin (renature), lc s cho php cc si n ca mu d bt cp vi cc si n ca mu ADN ban u (target ADN). Mc lai s ph thuc vo tnh c hiu (s tng ng) gia mu d v mu gc cng nh iu kin cho php lai thc hin (nhit , nng mui, pH, t ln mu d, kch thc mu d). Nh vy, vic chn iu kin chnh xc cho php lai c ngha rt quan trng cho tnh c hiu ca kt qu php lai. Sau khi lai, bc tip theo l ra bng m thch hp loi mu d d v cui cng l php o bc x nh du (tu theo phng php nh du phng x hay ho cht pht x hunh quang) nh gi mc tng ng ca php lai. Ni tm li mt s nhn t tham gia vo kt qu ca php lai l: mu d ADN, phng php nh du ADN, mu ADN lai v iu kin tin hnh v phng php nh gi kt qu php lai. + Chn mu d: Vic chn mu d c ngha quan trng cho php lai. Nhn chung cc nh phn loi hc vi sinh vt thng khng trc tip thit k mu d. V mt ny chng ti a ra mt s ni dung ch yu v tiu ch chn mu d theo Stahl v Amann (1991) nh sau: Mu d s lai vi ADN ch (target) v khng lai vi cc ngun ADN khc c mt trong mu lai. Khi phn tch cc mu vi sinh vt vi nhau th mu d nn l on nucleotid c trng cho cc mu phn tch phn bit vi cc sinh vt khc. Tuy vic chn mu d cng mang tnh kinh nghim, mu d i khi khng cn phi l ton b gene m ch l mt on gene m ho cho mt c tnh c trng cho i tng nghin cu (c th l yu t khng nguyn b mt, c t hay mt gene chc nng no trn plasmid). Vi cch chn mu d c kch thc nh (14-40 bp), c thun li l cc mu d c tng hp nhn to v php lai thc hin nhanh (di 30 pht) trong khi vi cc mu d ln thi gian ko di hn (khong 16 gi). Hn ch ln nht i vi cc mu d nh l kh khn khi nh du v dn n gim tnh c hiu ca php lai. Mt cch thng c s dng l thit k cc mu d t cc chui ARN thng tin 16S. Cch chn c th cn c vo on ADN c mt trong cc i din mc di loi, trong loi hay thuc chi nghin cu. + Cc phng php nh du: Cc k thut nh du ADN c xem l lnh vc pht trin mnh nht trong sinh hc phn t. Ni chung k thut nh du c chia thnh k thut nh du trc tip v gin tip. Trong phng php trc tip th

phn nh du pht hin c gn vo acid nucleic. i vi phng php gin tip th c mt phn chc nng c gn vo acid nucleic v phn ny li c pht hin gin tip da vo protein bm c hiu c pht hin bng k thut min dch. Sau y l chi tit cc phng php nh du. nh du trc tip:

- Bng di y a ra cc loi c cht dng cho phng php nh du trc tip da vo vic nhn ADN c nh du ln s tng nhy so vi phng php nh du ch c thc hin vi cc mi n l. i vi cc phng php nh du trc tip th cc nhm to tn hiu c gn trc tip bng lin kt ho tr vi nucleic ca mu d. - nh du trc tip bao gm 2 bc chnh: to tn hiu da vo lin kt ho tr ca nhm tn hiu vi mu d v thc hin php lai gia mu nghin cu v mu d. Bng 1.4. Mt s c cht dng cho nh du trc tip. Ngun to tn hiu 32 P
35

Ti liu Southern (1975)

Collins& Hunsaker (1985) Renz&Kurz (1984) Albarella & ADNerson(1986) Amman & ctv (1988) Urdea ctv(1988) Heller& Shneider(1983) Draper (1984)

Alkaline phophatase Ethidium Fluorescein Horseradish peroxidase Microperoxidase Nitrobenzofuran

Tetramethylorhodamine

Amman&ctv(1990)

Hnh 1.3. Minh ho phng php nh du trc tip (A) v gin tip (B) nh du gin tip:

nh du gin tip c thc hin theo 3 bc: thc hin php lai gia mu d v mu ADN ch, phn ng gia nhm chc nng v protein bm c hiu vi nhm chc tn hiu thng bo (reporter) v cui cng l to ra tn hiu t nhm chc tn hiu.

Bng 1.5. Lit k mt s h thng nh du gin tip.

Nhm chc nng bm protein

Nhm tn hiu (reporter)

Ti liu dn
Leary et al (1982)

Biotin-dUTP/steptavidin Digoxigenein-dUTP/antidigoxigenein Sulphone/antisulphone Dinitrophenyl/antinitrophenyl Poly(dA)/poly(dT)

Alkaline phosphatase Alkaline phosphatase Europium chelate Piruvate kinase Horseradish peroxidase

Kessler et al (1990) Syvanen et al (1986) Leller et al (1990) Morrisey & Collins (1989)

Acetyllaminofluorene/antiacetylaminofluorene

Alkaline phosphatase

Tchen et al (1984)

Mc d c nhiu h thng nh du v pht hin tn hiu nhng kinh nghim cho thy h thng biotin v digoxidenin cho nhy cao hn c. Hai h thng ny c th pht hin ti mc di picrogam acid nucleic. Trong trng hp vi biotin th i khi c mc nhiu nn cao do biotin c mt trong cc sinh phm, cn i vi digoxigenein th c th khng gp kh khn ny. + nh du phng x v ghi phng x: Phng php nh du phng x l phng php c dng ph bin trong cc phng th nghim lai ADN vi cc thnh phn nh du l 32 P v 35S. Kt qu ca php lai gia mu d v ADN ch c pht hin trn X-phim hoc nhp nhy lng. u im ni bt ca phng php nh du phng x l nhy c th t ti di mc picrogam ADN ch. Hn ch ca phng php ny l khng t c s thch hp cn thit cho cc th nghim chn on lin quan ti xc nh mu vi sinh vt, v d thi gian bn hu mu d ngn, nguy him cho ngi s dng v cn yu cu an ton nghim ngt cho phng th nghim v c nhn s dng cng nh vic qun l cht thi. Xut pht t thc t trn m cng ngy ngi ta cng quan tm n cc phng php nh du phi phng x. Mc tiu l t c mt h thng nh du c nhy tng ng vi phng php dng cht phng x. Bng di y lit k cc yu cu l tng cho phng php nh du mu d:

1, D gn vi acid nucleic theo phng php n gin v c lp li cao. 2, Bn trong iu kin lai v bo qun. 3, Khng b nh hng v lm nh hng n phn ng lai. 4, C th thc hin vi iu kin phn ng lai trong dch th (liquid phase) hay c cht mang (solid phase). 5, Phng php pht hin nhy v n gin. 6, D b loi b sau phn ng lai. 7, D phn bit gia mu lai v mu cha lai trong cng iu kin. 8, C th ng dng vi h thng t ng ho. Tuy nhin, nu theo cc yu cu l tng trn th cha c h thng nh du phi phng x no tho mn. Hin nay, c nhiu h thng nh du phi phng x t nhy cao v l l do cc phng php ny ang c ng dng rng ri c bit l phng php da trn cc enzym nh Alkaline phosphatase, Horseradisk peroxidase. Tuy nhin phng php s dng cc cht phng x hin ti vn ang c dng cho mt s phng th nghim c bit. Chin lc nh du vi cht phi phng x.

C 3 phng php nh du phi phng x: 1, Dng c cht ho pht x v sinh pht x, trong trng hp ny th c c cht ho v sinh pht quang u to ra cc bc x nh sng v sau l phng php pht hin bc x ny. * Vi ngun c cht ho pht quang c loi nh AMPPD (Bronstein, Edward & Voyta, 1989) c th c dng trc tip nh l c cht cho enzym Alkaline phosphatase trong php lai ADN t nhy cao trong thi gian ngn (Bronstein et al., 1990). * Phng php pht x s dng enzym Alkaline phosphatase trn c s gii phng D-luciferin t c cht, v d D-luciferin-O-phosphate (Miska v Geiger., 1987). Hai phng php ny tng i nhy v ang c s dng rng ri. 2, Phng php pht hin da vo thay i mu: Phng php o mu c th thc hin trn mi trng dch th hay cht mang v kh nhy so vi phng php pht quang. Ngi ta to ra

cc c cht sinh mu khi c mt enzym (chng hn nh Alkaline phosphatase). Li th ca phng php ny l vic pht hin n gin do kt qu l s thay i mu d pht hin v ng dng trong cc phng th nghim vi sinh vt. Mt phng php c th lm tng nhy ca phn ng mu bng cch thm mt enzym kch hot phn ng mu vo h thng. Mt v d cho iu ny l vai tr loi nhm phosphat ca phn t NADP thnh NAD do Alkaline phosphatase trong h thng pht hin mu d nucleic. Trong h thng ny th NAD c vai tr kch hot chu trnh oxy ho m c s tham gia ca alcohol dehydrogenease v diaphorase. Trong mi mt chu trnh th NAD s b kh thnh NADPH + H+. Phn ng oxy ho ny li km theo phn ng oxy ho m NADPH + H+ li b oxy ho thnh NAD, mi phn ng xy ra gn lin vi vic kh -indonitro-tetrazolium mu tm thnh formazan. Sn phm cui cng formazan c nh lng bng php so mu (Self. 1985). 3, C cht hunh quang v pht x hunh quang theo thi gian. S dng c cht pht x hunh quang l phng php kh nhy v c th pht hin c ti mt phn t cht pht x (fluorescein). Ni chung vi cc mu sinh phm thng c mc nhiu tn hiu nn cao. Thc t ny c th c ci thin vi c cht fluorophore bn b kch hot bi sng nh sng. S pht x sau c ghi li khi cc bc x nn b gim. i vi phng php dng Alkaline phosphatase th vic pht hin nhn t gn lanthanide theo thi gian i km vi hot tnh enzym ny gi l phng php pht quang lanthanide khuych i bi enzym (EALL: Evangelista, Pollack & Templeton, 1991). Trong trng hp ny, c cht khng c kh hnh thnh phc h gn vi lathanide pht x. Di tc dng ca Alkaline phosphatase th c cht v lanthalide b chuyn thnh phc h hot ng. Phng php ny kh nhy nhng hn ch ln nht ca n l cn thit b kch hot v o bc x hunh quang. + Qu trnh lai: Ni chung qu trnh lai (Hybridization ) c tin hnh theo mt trong 3 cch sau nh danh hay xc nh vi sinh vt: Lai vi vi sinh vt (in situ), lai trong dch th v lai vi cht mang (solid support). a. K thut lai vi vi sinh vt (in situ), hnh 1.4.

Hnh 1.4. Minh ho k thut lai in situ vi k thut FISH (fluorescence in situ hybridization) pht hin vi khun Helicobacter pylori. K thut ny c thc hin nh v chui acid nucleic trong vi sinh vt sau khi c c nh trong cc tiu bn hin vi. Tuy nhin, do hu ht cc vi sinh vt c kh nng thm (hay cho php) cc on ngn oligonucleotid nh du i vo t bo sau khi c nh mu v vy khi s dng mu d nh du vi cht nhum hunh quang c bit c th nhn thy c trc tip vi sinh vt di knh hin vi hunh quang. iu ng ch l phn ng lai phi tin hnh trong thit b c hn kn nhm hn ch vic bay hi ca dch lai. Vic bay hi dch lai ny dn n vic bm khng c hiu ca cht nhum hunh quang vi t bo. Nhit lai ph thuc vo oligonucleotid mu d. Sau phn ng lai th mu phi c xem ngay hoc c bo qun trong ti trong thi gian 6 thng. Nu mu d c hiu cho RNA th nhy tng ln rt ln v c ti hng ngn bn copy ca RNA trong mi t bo (Stahl v Amman, 1991). Lai trong mi trng dch th: Phn ng lai trong mi trng dch th xy ra nhanh hn nhiu so vi mi trng c cht mang pha rn (soild). Do c phn t ADN ch v mu d u c th di ng t do trong mi trng do phn ng t kt qu cao nht. Ni chung vi php lai thc hin trong dch th th thi gian kt thc nhanh hn t 5-10 ln so vi trong iu kin l cht mang (Bryan, et al., 1986). Tuy nhin, cng c th b sung thm cht mang lm tng phn ng lai. Cn thm bc cui cng l tch phc hp mu d-si ADN ch. Hn ch y l mu trong dch cho nn c th to ln cc nn km c hiu, do vy cn cc gii php lm hn ch mc nhiu ca nn cho thch hp.

Hnh 1.5. Minh ho k thut lai trong mi trng dch th. Trn thc t hu ht cc KIT thng phm dng cho vi sinh vt u tin hnh trong mi trng dch th. Vi sinh vt trong mu u tin c phn gii trong iu kin thch hp ADN ch lai vi mu d. Sau l bc lai v tch phc h mu d v ADN ch da vo cc thng s ca php lai theo kiu kp dp c th. Cc php lai theo kiu kp dp (hnh 1.9) cn c hai on ADN c trnh t khc nhau; mt on bm gi ADN ch gn vo cht mang v mt on l mu d. iu quan trng l hai on ADN ny phi c trnh t khc nhau cho d chng u gn vi hai vng khc nhau trn on ADN ch theo nguyn tc b sung. Mu d nh du c b sung vo trong dung dch c cc on ADN ch nghin cu. Nh vy theo hnh v phc hp ADN mu d nh du pht hin gn vi ADN ch v phc hp mu d- ADN ch gn vo cht mang c hnh thnh. Phc hp ny c tch ra khi dung dch v khi c mt c cht thch hp pht hin c mu d nh du. Khi dng h thng pht hin da vo cc ho cht hunh quang hay to mu c th dng thit b pht hin kt qu mt cch t ng. + K thut lai da vo mng: Tc gi u tin gii thiu k thut c nh ADN trn mng l Nygaard v Hall (1963, 1964). Sau chnh Southern (1975) l ngi m t vic tch ADN khi gel sau khi in di v chuyn chng ln mng nitrocellulose. Cc on ADN ch c pht hin bng k thut lai v y l bc tin quan trng ca sinh hc phn t. T nm 1977 n nay c nhiu ci tin v php lai cng nh bc chuyn ADN ln mng ca cc tc gi khc nhau nh: Meinkoth v Wahl (1984). Ngi ta cng s dng mt s mng nynol, mng nitrocellulose hot ho hay c bn cho cc php lai. i vi cng vic nh loi vi sinh vt vi k thut c nh trn

mng cho php lai ADN th cn chm mu (l ADN sch, hay t bo vi sinh vt hoc sinh phm c vi sinh vt nghin cu) ln mng thch hp. Mu c ly gii v ADN b bin tnh, sau ADN c c nh trn mng khi a vo t xy ti 80oC trong 2 gi hoc a vo x l vi tia UV trong trng hp s dng mng lynon. Mu d nh du sau c a vo dung dch. Bc tin lai c thc hin hn ch cc mu bm vo nhau khng c hiu. Sau l bc lai, mng sau khi lai c ra loi cc mu d cn d. Bc ra tip theo nh gi mc bm c hiu ca php lai. Sau cc mu d nh du lai vi v tr ADN mu trn mng c pht hin bng cc h thng pht hin tng thch. Ton b qu trnh lai trn mng c Meinkoth v Wahl (1984) m t chi tit. Khi pht hin typ vi sinh vt, chng ta phi s dng k thut Southern. Trong phng php ny ADN ca nhim sc th c tinh sch v c x l vi enzym ct hn ch thch hp, sau mu li c in di trn gel agarose v to ra du vn (finger print) ca nhim sc th. Sau khi in di gel c t di mng nitrocellulose hay mng nylon nm gia mt s lp giy. Lp giy lc pha trn c t trn cng phn gel v mng kp gia giy lc nh trong hnh 1.6.

Hnh 1.6. M t phng php chuyn ADN ln mng cho k thut Southern Blot. Kt qu l m t pha di c thm mt cch t nhin ln trn. iu gip cho vic chuyn cc mnh ADN t gel ln mng v bm cht vo mng vi kch thc v phin bn ng nh trn gel sau khi in di (m t chi tit theo Sambrook 1989). Phng php truyn thng ny cng c ci tin khi dng mng nylon chuyn ADN trong iu kin bin tnh (Read v Mann 1985). Thi gian chuyn c th vi gi hoc qua m. Vic c nh ADN trn mng c thc hin sau bng cch x l nhit hay tia UV nh trnh by trn. Tuy nhin, nhiu phng php ci tin c thc hin nhanh nh

chuyn bng in di (Bittner, Kupfere, Morris, 1980) hay s dng bm chn khng (Medveczky, 1987; Olszewsk, 1988). Trong nhiu trng hp dng in chuyn ADN t gel agarose ln mng th trc tin gel phi c x l bin tnh v c lm trung ho tr li. Gel c t gia hai bn in c cc bn giy thm (hnh 1.7).

Hnh 1.7. Minh ho bc chuyn ADN ln mng. Sau ngun in c ni v lc ADN s c chuyn ln mng. Thi gian chuyn s ph thuc vo kch thc on ADN nhng thng dao ng trong khong 1-3 gi. Phng php c th thc hin theo chiu thng ng hay chiu nm ngang. Hin nay c mt s thit b bn trn th trng c dng cho k thut ny. Vi thit b nm thng ng, th ton b c ngm trong m v c th tng cng dng in (ch v c th lm tng nhit ), phng php ny cn dng nhiu m. Ngc li theo phng php nm ngang hay phng php semi-dry, y gel v mng c kp gia cc bn giy lc tm t bng m thch hp. Trng hp ny cn rt t m v cng dng in l 1mA/cm2 l thch hp. Trong trng hp chuyn ADN ln mng bng p lc do chn khng, vic thit lp h thng c trnh by m gel c t trn mng v s dng lc ht chn khng a ADN ln mng. Dng lc ht chn khng t c hiu qu chuyn ADN ln mng cao hn phng php thm t nhin. Hiu qu ti a cho chuyn gel vi trng hp geneom sau x l enzym ct hn ch c th t c sau 1 gi. + Phn tch RELPs vi k thut lai ADN. Gii thiu phng php: Nh trnh by, s phc tp trong k thut RELP to ra nhng

kh khn cho vic xc nh cc tc nhn gy bnh trong cc nghin cu v dch t hc. iu ny cn kh khn v phc tp hn trong khi so snh cc kt qu thu c trn cc bn gel khc nhau hoc ti cc phng th nghim khc nhau. Nguyn nhn l do s lng cc bng ADN ln ti mc khng th xc nh kch thc ca chng hay cc bng thu c khng lp li cc kt qu nghin cu. Mc d vy, theo phng php ny cc ph ADN phc tp sau khi x l vi enzym ct hn ch s c lm bin tnh v chuyn ln mng nitroxenluloza hay nylon. Mng s c lai vi mu d thch hp, kt qu ph php lai s r v khng qu phc tp do n ch hin th cc mnh ADN bt cp vi mu d. Vi mt s lng khng ln cc mnh hin th thu c sau php lai s cho php xc nh c chnh xc hn v kch thc. iu ny lm c s cho so snh gia cc gel vi nhau cng nh kt qu thu c t cc phng th nghim khc nhau. Khi s dng phng php ny cn ch mt s mu d sau: 1, Mu d c th l on RNA ribosom t mt loi sinh vt no : v d rRNA ca E.coli c th c cc cng ty thng phm cung cp (Boeringer Mannheim), y l mu d kh thun li v c nh du phng x hay vi cc c cht hunh quang. u im chnh ca mu d ny ch phn RNA l on kh bo th do mu d c th dng lai vi cc sn phm x l enzym ct hn ch ca nhiu loi vi khun khc nhau. C mt s loi khi lai vi mu d ch cho mt kt qu ging nhau trong khi loi khc khi lai cc chng vi mu d th li thu c kt qu khc nhau. y l c s cho phng php ribotyping. 2, Mu d c th l on ADN ngu nhin c chc nng khng xc nh (Tompkins et al., 1986). Mu d ny thng c dng cho cc loi c cha chnh cc on ADN ny. S dng cc mu d ny i khi rt c ngha trong vic phn bit cc mu sau khi tin hnh phng php ribotyping (Saunders et al., 1990). 3. Mt cch khc na cng c dng l s dng mu d l mt on ADN tch dng t mt gene bit. V d dng on gene m ho cho c t ngoi bo (exotoxinA) s dng nh typ Pseudomonas aeruginosa (Ogle et al., 1987). Mu d dng gene m ho cho c t Cholera c dng xc nh cc typ cho cc chng thuc h phy khun sinh c t (Yam, Li Lung & Ng, 1989). Vic s dng bt k loi mu d no cng to ra ph c trng ca php lai c ngha cho so snh gia cc chng vi nhau. Hn ch chnh ca k thut ny ch kt qu thu nhn ch khu tr phn gene bt cp vi mu d m khng c trng cho c h gene. Bng 1.6. Kt qu thu c khi thc hin k thut RFLP vi dng cc mu d khng phi l ribosom.

i tng Aeromonas spp Borrelia burgorferi Candida albicans Chlamydia trachomatis Corynebacterium diphtheriae Cryptococcus noeformans Escherichia coli Hemophilus influenzae Histoplasma capsulatum Lactobacillus helveticus Mycobacterium tuberculosis Pseudomonas aeruginosa Salmonella spp Staphylococcus aureus

Tc gi nghin cu v ti liu dn Altwegg an Luthyhottenstein (1991) Wallich et al (1992) Schmid et al (1992) Scieux et al (1992) Groman et al (1993) Spitzer (1992) Bohm ADN Karch (1992) Forbes et al (1992) Leathet al (1992) Delostreyesgavilan et al (1992) Mazurek et al (1991) Tompkins(1991) Sodati ADN Piffaretti (1991) Goh et al (1992)

- K thut ribotyping: Nh trnh by trn mi mu d u cho cc kt qu c sc thuyt phc tuy nhin k thut dng mu d l on RNA ca ribosom a ra mt cch tip cn mi trong nghin cu dch t phn t vi cc vi khun c s khc bit ln trong khi cc mu d khc ch gii hn vi mt loi hay ch c ngha cho cc chng trong cng mt loi. K thut ny ln u tin c Grimont m t nm 1986 v nhanh chng tr thnh phng php hu hiu hin nay cho nghin cu dch t hc vi sinh vt mc phn t. Tnh hp l cho vic s dng k thut ny l ch gene m ho cho RNA ribosom c bo th cao. Cng c th pht hin thy nhng thay i cht t trong qu trnh tin ho i vi cc chui ADN trong cc vi khun nghin cu. Gene RNA ribosom c t chc thnh cc operon m cc gene ring r m cho cc RNA kch thc 5S, 16S v 23S chng c cch nhau bng cc on ADN spacer khng m cho gene no. Nu dng mu d hn hp gia 16S v 23S th kt qu php lai s hin th cc mnh tng ng vi phn ca gene ny trong khi dng mu d l on ca cc gene c tch dng c th a n kt qu hin th c phn gene tng ng v chui spacer. Nh vy s khc nhau ca kt qu ph thuc vo loi mu d s dng. Yu cu k thut:

bc thc hin. u tin l phi to ra c ph du vn tay thu c sau khi x l mu vi enzym ct hn ch. Ph vn tay ti u khi cc mnh ct phi c tch r v phn b ng u v kch thc trn gel. S lng cc mnh ADN thu c ph thuc vo mu ADN v loi enzym ct hn ch c s dng. Thng thng phi chn mt s mu x l th trc vi tng loi enzym (hay ng thi x l vi mt s enzym). C th thy r l cc enzym c 5 nucleotid ti v tr ct s to ra sn phm nhiu mnh hn cc enzym c 6 nucleotid ti v tr nhn bit. Thc t cng cho thy khi dng enzym ct hn ch (mt hay ng thi vi loi) thu c ph cc on ct hp l cho vic phn tch ribotyping th cc kt qu ny cng rt khc nhau. Nh vy, iu quan trng nn tin hnh trc cc php phn tch ny, phi chn c mt s chng c trng v th vi mt s enzym ct hn ch chn gii php cho nghin cu dch t hc vi k thut ny. Trong mt s trng hp kt qu phn tch khi ch tin hnh vi 1 hay 2 enzym ct hn ch c th khng a ra c kt qu chnh xc. Nh vy, khi c c kt qu hp l sau khi x l cc mu ADN vi enzym ct hn ch th tin hnh cc bc chuyn cc on ADN trn gel ln mng theo cc phng php c m t trn. Phng php chuyn bng chn khng l hp l hn c v kt qu cho vic chuyn cc bng c kch thc gn nhau li c tch ra sc nt trn mng. Khi thc hin php lai nn ch nu nh dng ngun ARN ribosom ca mt chng no nh du lm mu d th cn phi thay i iu kin lai nh nhit php lai c thc hin tt vi cc on ADN t cc chng vi sinh vt khc nhau. C mt s phng php nh du mu d l ADN ribosom. Phng php nh du phng x (Grimont, 1986), y l phng php c u th l ch cn vi bc n gin cho php lai v ra mu nhng li mang nhiu hn ch nh: thi gian bn hu ngn, yu cu an ton cho phng th nghim ring bit, nguy him cho ngi dng v gp kh khn vi vic x l cht thi phng x. Mi y c mt s phng php nh du mu d phi phng x c s dng kh thnh cng. Pitcher (1987) m t phng php tng hp mu d gn vi bilatin pht hin ARN ribosom ca Providencia stuartii. Cht kch hot quang ho c Koblavi v Grimont m t (1990). ARN ribosom cng c nh du bng acetylaminofluorence (AAF) do Grimont (1989) m t. Cng ty Eurogeneetik (B) cung cp phc hp AAF-rARN trn th trng. Gn y Gustafero v Persing (1992) a ra mt phng php mi m y phc hp horseradisk-peroxysase-polyethyleneimine vi rARN v kch hot bng quang ho. Cc mu d c nh du bng cc cht phi phng x ny c th cho kt qu nhanh tng

hnh php lai cc mu ca cc chng khc nhau trong cng gel chuyn ln c th so snh vi nhau khi dng k thut ny. Trong trng hp xc nh chnh xc kt qu cc mnh lai vi mu d c th tin hnh so snh cc kt qu thu c t cc phng th nghim khc nhau. + ng dng k thut ribotyping: Xc nh typ vi khun bng k thut ribotyping c mt s u im so vi mt s k thut nh typ khc ch: Thc t l cc gene ca ribosom kh bn do chng nm trn nhim sc th. Hu ht cc vi sinh vt u cha nhiu phin bn ca operon ribosom do khi lai vi mu d thch hp th kt qu l to ra mt s mnh lai c kch thc khc nhau. Hin nay, ngun rARN ca E.coli nh du l mu d kh ph bin c thng mi ho.

Hnh 1.8. Kt qu ribotyping vi Helicobacter pylori. Do tnh a dng v phc tp ca k thut nh typ theo ribosom do trn thc t khi nhn kt qu bng mt thng kh c th pht hin c s khc nhau hay ging nhau gia cc chng trong cng mt loi khi tin hnh nghin cu dch t hc trn din rng. Owen v cng s (1992) nghin cu kh nng tr gip ca computer so snh kt qu thu c khi nghin cu cc chng Helicobacter pylori. Tng t nh vy Bialkowska-Hobranska v cng s (1990) dng thit b laze o mt cc mnh ADN lai cng vi s tr gip ca computer phn tch cc kt qu thu c t cc chng cu khun nha bo gram m. Phng php ny c th a ra s liu chung lm c s cho xc nh s ging nhau gia cc loi khc nhau. Cc phn tch thu c t cc s liu ca cc cc th khc nhau c th ch ra c cc c th mi lm c s cho nh danh cng nh theo di.

Mi nghin cu cho thy mt iu quan trng v c ngha nht l cch chn enzym ct hn ch v loi mu d dng cho php lai (Saunder v cng s 1991). Tm tt: Nhn chung phng php lai ADN chng t u th ca n nh l mt k thut quan trng cho nh typ v nghin cu dch t hc nhiu loi vi sinh vt khc nhau. V nguyn tc phng php ny cng c u im v nhc im ca n. u im: - S dng cho nhiu i tng vi sinh vt. - C cc mu d vn nng c bn rng ri trn th trng. Kt qu lai c lp li cao v kh n gin cho vic phn tch kt qu. - C th s dng s tr gip ca computer lu gi v phn tch kt qu th nghim. Hn ch: - Phng php s dng kh phc tp v tn thi gian. - Thng tin kt qu ch phn nh cho on gene lai vi mu d s dng. Hin nay, c th s dng cc on ADN tng hp lm mu d v iu ny thc t lm ci thin nhiu mt ca k thut lai nh: khi c ADN tng hp th khng phi thc hin cc k thut tch v tinh sch cc mnh ADN tch dng c th ln ADN plasmid. Mt khc khi lai vi mu d tng hp th phn ng tin hnh nhanh vi c hiu cao hn. Cho d k thut ny c s dng tt cho nhiu i tng vi sinh vt khc nhau nhng nm 1992 Heimberger v cng s tin hnh phn tch ADN c x l vi enzym ct hn ch trong trng xung in (PFGE), kt qu ny rt c ngha cho php phn tch su hn v phn bit cc chng vi sinh vt lin quan n s bng pht dch i vi cc chng vi sinh vt khc. Ribotyping v PFGE c chung nguyn tc da vo s phn b ca cc v tr enzym ct hn ch trn ADN ca ribosom. Tuy nhin, s khc bit ch ribotyping phn nh s phn b ca v tr cc enzym ct hn ch nm trong cc gene m ho cho rRNA hay nm trong vng nhim sc th c bo th cao, cn i vi PFGE phn nh s phn b ca cc enzym ct hn ch phn b trn ton b gene nghin cu. Nghin cu ca Prevost (1992) cho thy l k thut PFGE c th hiu qu hn k thut ribotyping i vi php phn tch cc chng Staphylococus aureus khng methicillin. 2.3. Nhn gene v k thut gii trnh t ADN.

K thut lai ADN c s dng rng ri cho cc nghin cu xc nh typ ca nhiu i tng vi sinh vt. Ngun ADN ch i khi ch c s lng t tr khi cc tin hnh nui cy vi sinh vt. Trong mt s trng hp vic nui cy khng th thc hin c vi nhiu loi vi sinh vt hay virt hoc trong cc trng hp khc cn thi gian di nui cy vi sinh vt c ADN cho cc k thut lai hay nh typ. Kh khn ny c khc phc bng cch lm tng ngun ADN ch mt cch nhanh chng bng k thut nhn gene PCR. a. K thut nhn gene PCR (Polymerase Chain Reaction) Vic nhn ADN cho php lm tng s lng gp hng triu hoc nhiu hn na i vi on ADN hay ARN nghin cu. C nhiu phng php khc nhau phn tch ngun ADN khuych i theo cch ny. Phng php n gin nht l s dng in di xc nh kch thc sn phm ca phn ng PCR. C th xc nh nhy v tnh c hiu cao hn nu sn phm PCR c lai vi mu d c hiu c nh du. Phng php a li thng tin nhiu nht l xc nh c trnh t ADN v ARN ca sn phm PCR. Vic xc nh c sai khc ca chui ADN s cho php xc nh v nh typ chnh xc nht cc i tng vi sinh vt nghin cu. Kt qu xc nh trnh t ADN cn cho php xc nh mi lin h tin ho v dch t hc ca cc chng vi sinh vt. - i nt v phn ng chui PCR: Phn ng chui PCR nhm khuych i ADN ln u tin c Saki (1985) gii thiu v y c coi l mt trong nhng tin b khoa hc quan trng nht v sinh hc trong thp k 80. Phn ng PCR s dng enzym chu nhit to ra nhiu phin bn theo hm s m. C th v d, ch cn 1 si ADN sau vi gi c th nhn ln 1011 phin bn ADN tng ng vi 100 ng ADN. Sau khi thc hin phn ng PCR th cn tin hnh mt s k thut xc nh sn phm, n gin nht l in di v xc nh kch thc ca sn phm PCR. Trong cc nghin cu chn on th kt qu ny rt c ngha, n chng t s c mt ca ADN ch trong mu nghin cu. C th nhn c tnh c hiu v nhy cao hn khi s dng k thut RFLP vi sn phm PCR hoc lai vi mu d c hiu. Tuy nhin, thng tin y nht vn l kt qu xc nh trnh t ADN ca sn phm PCR. Ngay t khi c gii thiu th PCR c xem l phng php a li kt qu kh nhy trong vic xc nh v pht hin vi sinh vt. Tnh u vit ca n th hin ch c th ch cn mt ht virus hay mt t bo vi khun no cng cho phn ng xy ra v khuych i ti mc c th pht hin c trong thi gian ngn. K thut PCR cn c dng nhn ADN t khun RNA sau khi c chuyn thnh cDNA sau phn ng phin m ngc (RT). Phng php ny nhanh chng c ch v tr ln ph bin cho cc nghin cu pht hin vi khun hay virus nng thp trong cc mu mi trng v bnh phm. V d vic pht hin mu mu nhim HIV c ngha l pht hin phn nh ADN ca virus trong h gene

ngi c kch thc gp 100 000 ln. Mt khc khi dng k thut min dch vi khng th th phi cn ti 8 ngy mi c p ng min dch trong mu, trong khi vi k thut PCR th ton b quy trnh pht hin ch mt vi gi (i vi ngun ADN hay RNA). Li th ca k thut PCR c tn dng cho vic chun b ADN lm mu d vi s lng ln cho cc php lai ADN trnh by trn. Cc mu d c th c nh du bng phng x hay phi phng x khi tin hnh phn ng khuych i. Mu d nh du vi k thut PCR c u th hn cc phng php nh du truyn thng bng k thut tng hp cc mch t gy (nick translation) hay nh du vi mi ngu nhin (rADN random primer labeling). Cc u th ny c th k n, c th nh l cn lng nh si ADN khun, c th tin hnh nh du vi cc mu d c kch thc ngn, chun b mu d khng cn tinh sch ADN khun. Bng 1.7. di y lit k mt s v d ng dng k thut PCR cho pht hin nhiu i tng vi sinh vt khc nhau. Rt nhiu cc kt qu nghin cu nh vy c trnh by trong cc tp ch chuyn ngnh nh: nh nh: nh nh: nh nh: Journal of Clinical Microbiology, Applied Environmental Microbiology. Bng 1.7. Mt s v d v ng dng k thut PCR trong xc nh vi sinh vt.
Vi sinh vt Ti liu

Acanthamoeba sp Bordetella pertussis Borrelia burgdorferi Brucella sp Candida albicans Carnobacterium spp Chlamydia trachomatis Clostridium difficile Coxiella burneti Cryptosporidium parvum Cytomegalovirus Dengue virus Epstein-barr virus

Vokin et al (1992) Houard et al (1989) Marconi ADN Garon (1992) Herman an Derider (1992) Miyakawa et al (1992) Brook et al (1992) Hayes et al (1992) Gumerlock et al (1991) Stein an Raoult (1992) Laxer et al (1991) Gozlan et al (1991) Henchal et al (1991) Samoszuk (1991)

Erwinia amylovora Escherichia coli Frankia spp Gaeumannomyces spp Helicobacter pylori Hepatitis C virus Human immunodeficiency virus (HIV) Influenza virus

Bereswill etal (1992) Jackson (1992) Simonet et al (1990) Henson (1992) Claton et al (1992) Okamoto et al (1992) Dawood et al (1992) Claas et al (1992) Merien et al (1992)

Leptospira spp Niederhauser et al (1992) Litsteria monocytogenees Robertson et al (1991) Luteovirus Hackel et al (1990) Mycobacterium leprae Altamirano et al (1992) Mycobacterium tuberculosis Maiden et al(1992) Neisseria meningitis Charlotte et al (1993) Papillomavirus McOmish et al (1993) Parvovirus Barker et al (1992) Plsmodium falciparum Olsson et al (1993) Pneumocystis carini Yang et al (1991) Polio virus Seal et al (1992a) Pseudomonas solanacearum Gage et al (1992) Rickettsia spp Taniguchi et al (1992) Rotavirus Rahn et al (1992) Salmonella spp Murakami et al (1991) Staphylococcus spp

Toxoplasma gondii Treponema pallidum Trichomonas vaginalis Trypanosoma cruzi Vibrio vulnificus Yersinia

Vanevan et al (1991) Grimprel et al (1991) Riley et al (1992) Breniere et al (1992) Hill et al (1991) Nakajima et al (1992)

Sau y l nhng nt chung nht ca k thut PCR: Nguyn tc c bn ca PCR: + Khi qut chung: PCR l phng php dng enzym khuych i chn lc mt on ADN theo lut s m. Phn ng nhn gene c thc hin vi enzym ADN chu nhit, hai mi tng hp v 4 loi deoxyribonucleic (dATP, dGTP, dCTP v dTTP). Mi mt chu k phn ng nhn gene gm 3 bc (xem hnh v minh ho): Bc 1 l bin tnh ADN, c tc dng tch hai si n t si khun xon kp. Bc 2 l bt cp hai mi vo hai si n ca khun. Bc 3 l ko di chui theo mi, chiu 5-3 vi qu trnh trng hp gn cc dNTP dc theo si khun to thnh phin bn mi theo nguyn tc b sung. Tnh c hiu ca phn ng c quy nh bi mi v s sao chp trc tip theo trnh t si khun gia hai mi. Nh vy, kt qu to ra hng triu phin bn si khun trong vi gi. Mi cho phn ng l cc on ADN c kch thc 20-30 nucleotid c thit k theo trnh t ca on gene cn khuych i da theo d liu c sn. Mi cp mi phi hon thnh chc nng ca mnh trong ton b phn ng, tc l chng phi bm c hiu vo ng v tr ring cho tng mi. S d nh vy l do nu chng khng bm vo v tr c hiu th khng th khuych i c on gene ch. Vi cc gene c bo th cao nh rADN th khi cp mi c thit k th chng c th nhn c gene t nhiu i tng. Ngc li, trong nhiu trng hp dng mi khng c hiu th c th nhn c cc sn phm PCR khng c hiu. Tuy nhin, c hai kt qu trn u c dng cho nh typ vi sinh vt. Th nghim phn ng PCR ln u tin dng mnh Klenow ca enzym ADN polymerase I t E.coli. Tuy nhin, enzym ny b bin tnh ti 94oC v nh vy cn phi b sung enzym mi sau mi chu k phn ng. n nay qu trnh ny c ci thin do dng enzym Taq ADN polymerase (Taq = Thermus aquaticus ) chu nhit. Enzym ny c tch ra t vi khun sng sui

nc nng. Tc xc tc cho phn ng ca enzym ny rt nhanh, c th t khong 8000 bp/pht ti 755oC. Hot tnh enzym gim mt na khi x l ti 92.5oC trong 230 pht hoc 40 pht ti 95oC. Nh vy khi dng enzym ny th khng cn b sung enzym mi sau mi chu k phn ng. + Cc thng s k thut: Bt k mt phn ng PCR no cng bt u bng vic tch ADN lm si khun. Ni chung, theo l thuyt ch cn mt si khun cng cho phn ng to ra sn phm nhng trong thc t cn khong 10-100 ng/phn ng. i khi chun b ADN theo phng php n gin khng cn tinh sch cng ph hp cho phn ng PCR. Tuy nhin, iu quan trng l trnh s c mt ca EDTA (acide thylne-diamine-ttraactique) hoc m phosphat v kt qu s lm gim Mg++ (do EDTA hoc to thnh mui Mg3(PO4)2 khi nhit cao). Phn ng PCR c th nhn c on gene di 10 Kb nhng trong thc t thng dng nhn cc on c kch thc ngn hn (<2 Kb). C th tham kho cc thng s thnh phn phn ng PCR trong bng di y. Bng 1.8. Thnh phn phn ng PCR. Thnh phn ADN khun dNTP (mi loi) Mi xui Mi ngc Taq polymerase KCL Tris HCL (pH 8.4) ti 25oC MgCl2 Gelatin Nonidet-40 S lng 10-100 ng 200 mM 0.1 1.0 mM 0.1 1.0 mM 1U 50 mM 10 mM 2.85 mM 100 mg/l 0.05%

S chu k cho phn ng to sn phm c hiu t 20-30 chu k. iu ch l cc mi cho phn ng phi c nhit bt cp vi ADN ch gn nhau, c trnh t khng b sung nhau trnh bt cp vi nhau. Trnh t u 3 ca cc mi cng phi khc vi trnh t vng trung tm ca sn phm trnh to thnh hin tng kp tc. Ni chung, ngy nay ngi ta c th ti u ho vic chn mi nh s tr gip ca cc chng trnh my tnh. i khi cng xut hin hin tng nhn sn phm PCR khng c hiu, iu ny thng hay xut hin khi dng cc cp mi suy bin t chui acid amin. Tuy nhin trong hu ht cc trng hp ny th vic thay i iu kin phn ng c th hn ch c cc sn phm khng mong mun. Cc thnh phn nh nhit , nng Mg++ v enzym c nh hng ln n tnh c hiu. S c mt ca nonidet-40 c tc dng tng hiu sut ca phn ng.

Vic ng dng k thut RFLP vi sn phm PCR c th l cch nhanh chng tm s khc bit i vi cc gene khc nhau. Vi cch ny yu cu sn phm PCR phi sch v ng nht. Theo cch ny trnh t c th c thc hin nh sau: Sau khi in di kim tra sch ca sn phm PCR, tin hnh kt ta vi ethanol. Kt ta c ho vi m thch hp v sau c x l vi enzym ct hn ch thch hp. Kim tra kt qu thng thng trn gel agarose, trong mt s trng hp c th trn gel polyacylamid thu c phn gii cao hn. Ch rng thng thng hot tnh enzym gim 5% sau mi chu k nhit. Nh vy c th c lng 50% hiu qu khuych i mt i trong ton b qu trnh phn ng. + Mt s kh khn vi phn ng PCR:
PCR l k thut c ngha ln trong nghin cu sinh hc phn t nhng cng ny sinh mt s vn cn ch c bit khi pht hin mt s bnh virus hay vi khun. Vn y l acid nucleic ca c t bo cht v t bo sng u cho kt qu dng tnh trong phn ng PCR. Phn ng nhy nn mi s nhim ADN u c th a n sn phm dng tnh gi trong kt qu. hn ch thc t ny cn tin hnh vi cc mu i chng cn thit bit c s nhim tp t cc thnh phn phn ng hay t dng c th nghim. Mt vn na cng cn cp l sai s trong phn ng nhn gene vi enzym taq ADN polymerase. Sai st thm nucleotid xy ra vi mi mt n v sao chp khong 9000 nucleotid v t bin dch khung xy ra vi s lng tng ng khong 40 000 nucleotid.

+ Mt s k thut PCR khc: Mt s k thut pht trin trn c s ca k thut PCR c ngha quan trng cho nh danh v nh typ vi sinh vt l nested PCR (PCR lng), revert transtriptase PCR (sao chp ngc) v asymmetric PCR (PCR mt chiu). PCR lng l phn ng PCR bao bm hai phn ng ng thi, phn ng u vng ngoi gene v sn phm ca n li lm khun cho phn ng sau cho on nm trong gene. Nh vy phn ng sau cn 1 hay 2 mi nhn phn gene nm trong sn phm PCR t hai mi pha ngoi to ra. Vai tr k thut ny lm tng nhy v c hiu ca phn ng PCR. Phn ng PCR phin m ngc c dng nhn ADN t ARN ca virus. Vic kt hp phn ng ny vi PCR lng c tc dng pht hin ARN ti thi im nht nh ca mu. K thut PCR mt chiu c ng dng trong phng php gii trnh t ADN trc tip t sn phm PCR.phm PCR. + S dng k thut PCR cho xc nh typ vi sinh vt: Nh trnh by trn, dng cp mi c hiu c th xc nh c gene c trng cho loi hay thm ch mt chng vi sinh vt no . Cc

chin lc ng dng li th ca k thut PCR dng trong xc nh typ vi sinh vt trong nghin cu dch t hc c chia lm 4 nhm sau: - Phn tch RFLPs vi sn phm PCR khi dng cp mi c hiu.
Trong trng hp ny dng kt hp k thut PCR v x l sn phm PCR vi enzym ct hn ch v quan st kt qu sau khi in di sn phm trn gel agarose hoc polyacrylamid Mt dn chng cho k thut ny l kt qu nhn gene synthase citrat trong Ricketsiae sau sn phm PCR c x l vi enzym ct hn ch phn bit gia cc chng vi nhau (Regnery v Ctv, 1991). Cc v d sau (bng 1.8) cho thy hiu qu ca phng php ny.

Bng 1.9. Mt s v d cho k thut RFLPs (Restriction Fragment Length Polymorphism) phn tch cc gene c hiu c nhn ln bng k thut PCR. Chng vi sinh vt nghin cu Chlamydia spp. Clostridium spp. Helicobacter pylori Hepatilis C virus Mycobacterium tuberculosis Nitrobacter spp. Rickettsia spp. Streptococcus spp. Tc gi nghin cu v ti liu dn Kaltenboek et al (1992) Gurtler et al. (1991) Foxall et al. (1992) Okamoto et al.(1992) Ross &Dwyer(1993) Navarro et al.(1992) Regnery et al. (1991) McClellADN et al. (1992)

+ Dng k thut PCR cho ribotyping: K thut PCR ribotyping l ng dng cc cp mi c hiu nhn cc vng gene ca ARN (thuc ribosom hay ARN vn chuyn). Tuy nhin, trong thc t k thut ny yu cu kinh nghim, k thut v tn thi gian thc hin php lai. khc phc hn ch ny mt cch tip cn c trnh by y l phn tch a hnh (du vn tay) ca vng gene ny hay vng gene xen k ca rARN hoc tARN. Hu ht cc chng vi khun u cha 2-22 phin bn rARN trong mi t bo (Gottlie v Rundner, 1985) trong khi vng xen k gia 16S v 23S cha mt s cc on ADN lp li (Bacot v Reeves, 1991). Trong khi on gene m ho cho tARN kh bo th th vng xen k ca tARN li thay i t 2-35bp i vi Bacillus subtilis (Vod, 1985) v t 2-208bp trn i tng E.coli (Jinks-Roberson v Nomura, 1987). Nh vy khi s dng cp mi nhn ton b vng gene tARN c th to ra c s khc bit gia cc chng vi sinh vt. Kt qu ng dng k thut ny trn mt s i tng c trnh by trong bng 1.9.

Bng 1.10.. Mt s v d s dng k thut PCR cho ribotyping.

Vi sinh vt Vi khun lactic Listeria Mycoplasma Pseudomonas cepacia Pseudomonas solanacearum Staphylococcus spp. Streptococcus spp.

Tc gi v ti liu dn Klijin etal.(1991) Czajka et al.(1993) Van Kuppeveld et al.(1992) Kostman et al.(1992) Seal et al.(1992b) Welsh &McClellADN(1992) McClellADN(1992)

Khi s phin bn rARN tng c ngha l lm tng nhy ca phng php ng dng. Engstrand v cng tc vin (1920) cng b c th s dng mu d l on nucleotid b sung vi on 16S ca rARN nh l mt trong s mi cho php nhn phin m ngc ARN v php phn tch c thc hin nh k thut PCR nhn sn phm cDNA c dng cho pht hin Helicobacter spp. nhy ca phng php pht hin ny tng 50 ln so vi phng php truyn thng. + K thut rep-PCR: Mt phng php trc tip cho kt qu finger printing khng cn s dng enzym ct hn ch l rep-PCR da trn nguyn tc nhn cc on gene lp li. Thut ng rep-PCR l ch phng php chung s dng cp mi c hiu nhn cc chui lp li ph bin trong cc vi sinh vt thuc c th nhn s. Cc chui lp li c bo th cao trong cc i tng thuc Enterobacteriaceae v mt s i tng khc (Versalovic v Lupsky, 1991). Dng cc on mu d bo th c th lai vi c hai on ADN lp li: on c kch thc 126bp (Enterobacterial repetitive intergeneic concensusERIC) v on 38bp (repetitive extrageneic palindromic-REP) t b gene ca Enterobacteriaceae vi khun gram m v mt s chng c quan h xa khc. C th trc tip thit k cp mi cho cc on gene bo th thu c kt qu finger printing sau khi nhn gene dng b gene ca cc vi sinh vt c mang cc on gene bo th ny. Sau khi in di c th pht hin c trn agarose cc on gene c kch thc khc nhau t cc ngun vi sinh vt khc nhau. K thut ny c ng dng em li kt qu tt khi nghin cu mi quan h trn cc i tng Bacillus subtilis (Versalovic v cng s, 1992), Citrobacter diversus (Woods v cng s, 1991), Rhizobium meliloti (Brujin, 1992). Thc t phng php to ra kt qu finger printing a dng t hu ht cc i din ca Enterobacteriaceae (Versalovic v cng s, 1991) v c ng dng trn tt cc vi khun c mang cc on gene bo th lp li trn. Phng php tng t cng cho php pht hin s khc nhau t cc ngun ADN trn cc i tng nhn thc thuc chi Naegleria nh m t ca Belkim v Quint (1992). Phng php ny dng cho pht hin a hnh cc on gene chung cho c vi sinh vt nhn s v nhn chun. + K thut RAPD (Random amplified of polymorphic DNA):

Hn ch ca cc phng php nghin cu trc y l phi dng cp mi c hiu. Theo cch ny phi c cc thng tin lin quan n on gene nht nh v i tng nghin cu. Kh khn ny c loi b trong trng hp dng cc mi ngu nhin hay tu tin, nh vy phng php phn tch khng cn n thng tin v geneome ca i tng nghin cuC s ca phng php ny c Welsh v McClell ADN (1990) m t khi dng mi ngu nhin nhn gene v to ra ph finger printing c trng cho tng h gene (geneome). Mi ngu nhin c th ch gm 5 bp nhn gene c th to ra kt qu finger printing kh a dng. C th xem kt qu minh ho trong bng v hnh sau. Bng 1.11. V d minh ho cho k thut RAPD. i tng Agaricus bisporus Aspergillus fumigatus Bacillus thuringiensis Brucella spp. Campylobacter spp. CADNida spp. Casuarina spp. Clostridium difficile Frankia spp Fusarium solani Helicobacter pylori Histoplasma capsulatum Lactococcus lactic Leptosphaeria maculans Listeria monocytogenees Porphyromonas gingivalis Rhizobium spp. Staphylococcos spp. Streptococcus spp. Streptococcus uberis Tc gi v ti liu dn Khush&ctv(1992) Aufauvre-Brown (1992) Brousseau&ctv (1993) Fekete&ctv(1992) Mazurier&ctv(1992) Lehmann 7 ctv (1992) Sellstedt& ctv(1992) McMillan& Muldrow(1992) Sellstedt& ctv(1992) Crowhurst & ctv(1991) Akopyanz & ctv(1992) Woods& ctv (1993) Cancilla & ctv (1992) Goodwin&Annis(1991) Czajka & ctv (1993) Menard &ctv (1992) Harrison &ctv (1992) Weish &McClellADN (1990) Weish &McClellADN (1990) Jayarao &ctv (1992)

Hnh 1.9. Minh ho kt qu phn tch s dng k thut RADP. b. Gii trnh t ADN Phng php chnh xc v a li nhiu thng tin nht cho nh danh v nh typ vi sinh vt l xc nh chnh xc trnh t chui ADN ca mt vng quy nh trn nhim sc th. Phng php gii trnh t ADN pht trin nhanh chng, kt qu so snh s tng ng ca trnh t gene nghin cu tr thnh phng php phn loi chun theo sinh hc phn t trong nghin cu h thng hc v cy ph h di truyn gia cc i tng nghin cu. Cc gene bo th c gii trnh t lm c s xc nh v tr ca sinh vt nghin cu, trong khi cc trnh t khng tng ng (khc bit) li lm c s cho s bit ha cng nh xc nh mi quan h gia cc c th gn vi nhau. + Nguyn tc: Phng php gii trnh t ADN theo nguyn tc to ra cc mnh ADN c nh du ti u 5 hoc 3. Cc mnh c cc base nh u c tch ra trn gel polyacrylamid Cc mnh c phn bit v v tr trn gel vi s sai khc ch vi mt nucleotid. Vic nh du v c kt qu theo cc k thut v phng php khc nhau. Vic to ra cc mnh ADN sai khc nhau v 1 nucleotid c trnh by theo 2 phng php: phng php ha hc (Maxam & Gilbert, 1997) v phng php enzym kt thc phn ng chui (Sanger, 1997). Ngy nay phng php enzym c dng ph bin hn c. + Phng php enzym kt thc phn ng chui ca Sanger: Phng php kt thc phn ng chui c thc hin da trn vic nhn ADN t si khun khi c on ADN mi vi xc tc ca Klenow hay T7-ADN polymerase v 4 loi deoxyribonucleotid (dNTPs). Nh vy, c 4

phn ng c tin hnh ng thi v trong mi phn ng th c mt loi dNTP b thay i bi mt dn xut lm dng phn ng (ddNTP). Kt qu l phn ng chui b dng c hiu cho mi mt base xc nh xy ra mt cch ngu nhin trong mi phn ng. Trnh t ca ADN c xc nh trn gel. u tin phng php ny c xy dng cho xc nh trnh t si ADN n to ra bng cch tch dng ADN vo vect thc khun th M13 (Sanger v cng s, 1978). Sau phng php xc nh trnh t si i ADN c Chen, William (1986) m t khi tch dng ADN vo plasmid. Ngy nay ngi ta la chn phng php Sanger gii trnh t ADN cho nghin cu phn loi v xc nh typ vi sinh vt thay cho phng php ha hc. Mt s li th ca phng php ny ch: Phng php ny n gin v khng tn nhiu thi gian nh phng php ha hc. Khi nghin cu phn loi v xc nh typ th cc gene nghin cu c bo th nht nh v vy ta c th dng chung mi cho nhiu i tng khc nhau. Mt trong hn ch ca phng php ny ch cc on ADN cn c tch dng trc khi tin hnh gii trnh t ADN. Tuy nhin cho n nay ngi ta ci tin phng php ny c th gii trnh t trc tip sn phm PCR. + Gii thiu k thut gii trnh t ADN theo Sanger: C hai phng php: Phng php gii trnh t truyn thng c kt qu trn bn gel (nh du bng phng x l ch yu) v phng php gii trnh t v c kt qu t ng (nh du bng ha cht hunh quang). - Phng php truyn thng: Cc bc chnh cho gii trnh t ADN nh sau: Chun b ADN khun Bt cp ADN khun v mi Tin hnh phn ng gii trnh t ADN Tch sn phm trn gel polyacrylamid bin tnh vi urea. c kt qu. Tuy nhin cn ch mt s im sau: 1. 2. 3. 4. 5. Chun b ADN khun. iu kin bt cp mi. Thc hin phn ng gii trnh t ADN. Tch sn phm trn acrylamid gel. c kt qu.

Mt s im cn c chi tit thm cho mi bc nh sau:

1, Chun b ADN khun: Si khun cn c hiu l vic gii trnh t c thc hin vi si n (vect M13) hay si i ADN. Vic c trnh t theo si n thng cho kt qu l kch thc on gene c di v chnh xc cao. Ngy nay ngi ta thng c si i v vic chun b mu d dng hn, ngoi ra khi s dng hai phn ng vi hai mi tng thch th qu trnh c li chnh xc hn. l u th ca cch c si i ADN. Do vy, rt cn c kt qu t c hai si ADN. Hin nay phng php c trnh t ADN trc tip t sn phm PCR ang ngy cng tr ln ph bin. 2, Chun b si ADN mi cho bc bt cp vo si khun: Ngi ta c th dng cc on ADN mi tiu chun c hiu cho trnh t ADN ca vect gn st vi si khun hay primer c hiu ca si khun m n s bt cp vo. Tin ch ca vic dng mi chun ch mi iu kin c la chn cho si khun v mi c dng lp i lp li nhiu ln cho cc si khun khc nhau. Hn ch ca loi mi ny ch ch c c mt on ngn ca gene (300 500 bps) trong trng hp phi c cc gene c kch thc ln cn phi tin hnh subclone c tip. iu ny c th thc hin bng cch to ra cc on ADN ngu nhin t ton b gene sau li gn vo vect c cho ht trnh t ca gene. Phng php tip theo l s dng mi c thit k theo nguyn tc b sung vi trnh t ca gene. Theo cch ny, ln lt cc on c c m khng cn thao tc subclone. Tuy nhin theo cch on mi c thit k c gene nh vy th iu kin cho tng phn ng gii trnh t ADN vi cc mi khc nhau s khng c ti u. Tuy vy, hin nay vic tng hp mi tr nn d dng v ph bin vi gi thnh thp, hn th na vi s tr gip ca tin hc th bc thit k mi c ti u ho. V vy phng php ny ngy cng tr ln thng dng hn. Sau khi mi c thit k th vic tip theo l chn cch nh du vo mi hay vo on ADN s c tng hp. u th ca vic nh du ngay ti u 5 ca mi ch cc bng ADN thng c cng mt cng tn hiu. Thng thng vi phng php c trnh t th cng th P32 thng c dng. u th ca n l mang gc photphat cao nng bta mnh, do ch cn trong thi gian ngn l thu c tn hiu. Hn ch ca phng php ny l do mang nng lng cao nn kh phn bit gia cc bng khc nhau so vi dng S35. Hin nay gii quyt vn ny ngi ta dng P33 tuy gi thnh cao nhng cho kt qu tt hn. 3, Thc hin phn ng gii trnh t ADN: Enzym thng dng thng dng l sequenase (l mt dng ca T7 DNA polymerase), enzym ny c u th hn hn so vi Klenow c dng trc y. Phn ng c thc hin nhanh hn v thu c kt qu ti u nu c mt ion Mn++, ion ny gip cho qu trnh sao chp c chnh xc cao. Trc y dNTPs nh du vi P32 c dng nh du sn phm.

Tuy nhin ngy nay nhiu ngi chuyn sang dng S35 thay th cho P32, kt qu l cho phn gii cao hn. Mi y phng php gii trnh t da trn k thut PCR c gi l chu k gii trnh t trc tip ADN ang thay th phng php c dng enzym sequenase. V nguyn tc th cc phng php ny l ging nhau. u th ca phng php ny l: lng ADN i hi t v khng yu cu phi c mc tinh sch cao. Ngoi ra, c mt u im na l phn ng xy ra nhit cao, do khc phc c nhng hn ch ca cc phng php khc khi gii trnh t i vi cc si khun c cu trc bc 2 (Fan & cng s, tp ch Biotechniques 21: 1132-1137). 4, Tch sn phm trn gel urea-acrylamid: Cho n nay c nhiu tin b trong k thut in di gel k t nm 1970 khi m ngi ta pht hin ra phng php ny. Tin b quan trng nht l gel gradient to ra dn cch thch hp c c thm t 50100 base. u tin, gradient c to ra gia acrylamid v gradient mui khi gel. C th c cch khc na l ngi ta thay i dy ca gel bng spacer. Phng php d nht l b sung nng mui NaOAc vo b m di khi chy gel, kt qu s to ra mt gradient cn thit m khng my kh khn. C nhiu cch ci tin khc nhau to ra gel c phn gii cao, v d to ra gradient i vi acrylamid mch thng hay b sung mt s cht to ra gradient (Long Ranger). Mt khc vic gel cng c n gin ha bc s dng bng dn. Mt s ngi dng cc gel siu mng t phn gii cao. Nhiu thit b c pht minh nhm thu nhn cc bng c tch ring khi chng ra khi gel, v gn y nhng thit b ny c thng mi ha. Nhum bc l phng php tng i hiu qu xc nh cc trnh t ADN, cch ny hn ch c nhiu bc dng trong cc k thut da vo nh bc x m khng cn s dng cht phng x. 5, c trnh t gene: Hin nay k thut c phim t ng thay th cng vic c trnh t ADN bun t trc y. C hng lot cc thit b t loi c th ghi li trnh t da vo vic chn tng loi base n nhng thit b c th thc hin c ton b qu trnh ny mt cch t ng. Hy vng rng vi nhng bn lun ngn gn ny s gip bn lm quen dn vi nhiu loi k thut khc nhau. Ngy nay cc thit b gii trnh t t ng cng tr ln thng dng v vic s dng loi thit b v ha cht khc nhau rt a dng. Vic chn la thit b, ha cht v phng php thch hp gii trnh t ADN nhanh, chnh xc v kinh t l iu cn c quan tm n.

* Mt s phng php c th thng c dng cho k thut gii trnh t ADN. Phng php 1: Tinh sch Plasmid cho gii trnh t ADN. Hin nay c nhiu k thut tinh sch plasmid khc nhau, phng php c trnh by di y l phng php thu c plasmid c tinh sch cao, c th dng cho c gii trnh t ADN th cng v t ng ha. Qu trnh ny l ci tin phng php s dng dch kim lm tan t bo, sau s dng sc k qua ct trao i ion. Cng nh cc sn phm c lu hnh trn th trng ca cc cng ty, hn ch ln nht ca phng php ny l gi thnh ca nha trao i ion tng i t. Thc t c rt nhiu cng vic phi gii trnh t mt cch th cng v cc cn b nghin cu tm ra nhiu phng php tinh sch khc nhau thu nhn ADN t sch cn thit, thm ch c th dng cho gii trnh t ADN t ng. Tuy vy, nhiu ngi vn thch s dng ct trao i ion, kt qu c th c c ti 600 bps hoc nhiu hn na. Qu trnh tinh sch nh sau: + Nui cy vi khun mang plasmid trn mi trng chn lc vi khng sinh tng ng (khong 3 ml). 1, Phn 3 ml mi trng LB cha 100 mg/ml apicilin trong ng nghim v trng (12X100 nm c nt). 2, V trng u que cy trn la v lm ngui trn mt a thch. 3, Ly sinh khi t mt khun lc v a vo ng nghim cha mi trng nui cy trn. 4, Nui cy vi khun 37oC qua m c lc va phi. + Chun b ADN dng ct Qiagene: Cc ct Qiagene c chun b d dng v nhanh tinh sch ADN khng cn dng gradient CsCl. ADN c chun b theo phng php ny t kt qu rt tt cho vic bin np v gii trnh t gene.
C hai ch quan trng khi dng ct Quiagene l: (1) Khng s dng qu nhiu mu ln ct, iu ny rt quan trng v nu dng th tch qu nhiu kt qu l s c nhiu protein nh hng n kh nng bm ct ca ADN. Vi cch ny c th nhn c lng ADN c cht lng cao hn t th tch t hn. Nu nh lng ADN thu c t t ln th u tin th cn thc hin ng theo ch dn ln th th 2 vi th tch ln hn. Khi c mt lot th tch mu c Quiagene khuyn co ln ct t c hiu sut tinh sch cao nht th nn dng lng dch ln ct c th tch nh nht. (2) m bo ct phi c ra hai ln trc khi gii phng ADN ra khi ct, iu ny cng rt c ngha v vic ra nh vy s loi tr c protein v cc thnh phn tp nhim khc.

+ Cc bc dng ct trao i Qiagene (ti liu hng dn s dng Quiagene): 1. 2. 3. Ly tm khong 1,3 ml dch vi khun mang plasmid. Lm tan vi 0.3 ml m P1 c RNase, chuyn sang tube mi. Thm vo 0.3 ml m P2 v trn u, gi nhit phng 5 pht (khng nn lu hn). Thm vo 0.3 ml m P3 trn u ngay nhng phi nh nhng, ly tm 4oC trong 30 pht vi tc 15000 v/pht. Thu ly phn trn ta. Ly tm li nh bc 3 mt ln na loi cc phn cn nu c. Cn bng m cho ct Qiagene-tip 20 vi 1ml QBT v cho m chy ht t do. a phn dch thu c bc 4 ln ct v t chy qua ct. Ra ct 2 ln, mi ln 1 ml m QC. Thu ADN vi 0.8 ml m QF. Kt ta ADN vi 0.7 th tch isopropanol (trc a v nhit phng). Ly tm 15000 v/pht ti 4oC. Ra ADN thu c vi ethanol 70%, kh 5 pht v ho tan li trong th tch m thch hp (25ml). Ch trong trng hp mu dng cho gii trnh t ADN t ng hunh quang th ho tan mu trong nc.

4. 5. 6. 7. 8. 9. 10.

Ni chung s dng ct Qiagene thu c trn 90% ADN v thng c dng cho tinh sch Plasmid v cosmit (kch thc di 2 Kbs n ln hn 50 Kbs). S lng plasmid thu nhn c t t bo E.coli ph thuc vo h t bo ch, loi plasmid t thp trung bnh n s lng plamid cao v iu kin nui cy (c th tham kho ti liu lin quan t cc ch dn ca Qiagene quyt nh s lng t bo dng x l cho mi loi kch thc ct). Ch cch lm sch v ti s dng ct: 1, Thm ethanol y vo ct, ly tm. 2, B ethanol v ly tm li mt ln na. 3, Lp li bc 1 v 2 vi nc ct. 4, Gi ct trng thi kh cho n ln s dng v sau. (Ct c th c dng cho nhiu ln tuy nhin khi mun tinh sch ADN dng cho tch dng th nn dng ct mi).

Cc loi dch m dng cho ct Qiagene: m P1 m P2 m P3 m QBT m QC m QF 50 mM Tris -HCl, 10 mM EDTA, pH 8.0, Rnase A pha ngay trc khi dng trong m P1 nng 100 g/ml 200 mM NaOH,1%SDS 2,55M KAc, pH 4,8 750mM NaCl,50mM MOPS,15% ethanol, pH 7,0, 0,15% Triton X-100 1M NaCl, 50mM MOPS, 15% ethanol, pH 7,0 1,25M NaCl, 50mM MOPS, 15% ethanol, pH 8,2 Bo qun 4oC Nhit phng Nhit phng Nhit phng Nhit phng Nhit phng

Phng php 2. Chun b gel cho gii trnh t ADN Gii thiu chung: Phng php ny gii thiu cch gel, ln mu v chy gel gii trnh t ADN. C nhiu cch gel, c th tham kho cc cch khc nhau chn cch tin ch nht. iu quan trng nht l cc bn knh phi c ra sch tuyt i. 1, Mang gng tay v loi b ht bt gng tay i. Ra bn knh mt ln vi nc nng. Sau dng bn chi ra vi 0.5N NaOH. Lau bn knh vi dung dch Alconox hay cht ty ra c chc nng tng ng. Sau ra sch li bng nc. 2, Ra cc bn knh vi nc trao i ion sau dng ln gi cho kh, trnh bi bm vo knh. Lau vi ethanol v dng giy Kimwipes hay Scott Utinity lau cho sch khng to vt. Kim tra li bi bm vo knh lau cho sch, sau knh c lau bng lp dch Rain-X hay Sigmacott, Gel Slick ca hng AT Biochem. Mc ch dng cht ny l gip cho vic gel d dng hn v gel khng b dnh vo cc bn knh. 3, Ra li bn m cch gel (spacer) vi nc t vo dc theo hai cnh i xng ca bn knh. 4, Ghp hai bn knh vi nhau. t cn thn hai bn knh bng nhau sao cho hai bn spacer st tn cui bn gene (to mt phng cng vi hai bn knh). Nu khng m bo mt phng th d b chy gel trong qu trnh gel. Kp 2 cnh ca gel ti 3 v tr. 5, Chun b gel acrylamid 8% Long Ranger (Hoefer Rig). Trn u hn dch:

16 ml Acrylamid Long Ranger (LR) 50%. 5 ml 10X TBE 70 ml 10M Urea (46 gam) a n th tch 100 ml vi nc Cho vo 800 l 10% Ammonium persulphat. 50 l TEMED. Qu trnh polyme ha c thc hin trong 15 -20 pht. Chy gel LR trong di nhit 50-55oC, nhit cao hn 55oC s ph cc lin kt acrylamid LR gel l dng ci bin cho gel acrylamid c mt s u im sau: Th nht l LR gel vi nng cao hn nhng vic chy gel th cng nh gel c nng thp thng thng, v d LR gel 5% tng ng 4% gel acrylamid vi bisacrylamid Th 2 l LR gel c kh nng phn gii cao hn v chy vi tc nhanh hn 30-40% gel thng thng, gel LR kh nhanh hn v khng b dnh. Mt s ch khi thao tc:: a. b. c. Trn gel nh nhng (trnh trn mnh). Ht dch acrylamid bng pitpet hay c th dng cc ong. t bn knh nghing 15o v gel bng cc ong hay pipet. Kim sot tc gel bng gc nghing. Trnh dng gel khi ngt qung v iu ny s dn n to bt. Nu phi dng li ht dch mi vo syringer th lc phi h dn bn knh xung v tr nm ngang trc khi dng gel. Sau ly dch gel mi vo syringer v tip tc gel trc khi gel ln khi v tr nm ngang. C thao tc nh vy n khi y bn gel. Nu xut hin bt khi gel th phi dng li ngay v nghing tm knh cho bt kh i ln, c th g nh tay vo bn knh cho bt kh i ln ht. Phi loi ht bt kh ra khi gel, sau mi tip tc gel. 6, t gel nm ngang v a cnh bng ca lc rng c mp vo gel, to mt gc hi nghing nhm trnh to bt kh. Chc chn rng t cnh bng ca lc vo pha trn ca gel v phn rng c mp s nm dc theo nh ca bn knh di (lc s nm ngc vi v tr trong hnh 1.10). B sung thm gel v y thm lc vo ng v tr v c nh lc li bng kp trnh x dch. 7, Bao bc gel vi giy nilon SaranWrap trnh gel tip xc vi khng kh. Ngoi ra c th thm giy lc vi m TBE y ln. iu ny gi cho gel lun m v trnh gel tip xc vi oxy (oxy lm cn tr qu trnh polyme ha).

ha c th ht mt t gel vo pipt pastuer v cho n khi qu trnh polyme ha xy ra. Hoc c th ly gel cho vo ng falcon v y cht np li, trong nhiu trng hp th gel ch nn polyme ha trong 15-20 pht hoc nhanh hn. Nu qu trnh polyme ha khng xy ra th li phi tin hnh ra knh v li gel. Ch nhiu kh nng qu trnh polyme ha khng xy ra do Ammonium persulphat khng cn tt khi dng lu, nn chun b dch ny dng trong tun. iu cn ch na l gel phi c bc k vi SaranWrap qua m. 9, Chun b gel trc khi chy: a. Ly SaranWrap ra khi gel. b. t gel vo thit b in di (bn knh di t p vo thit b), chc chn l ct b lp bng dnh y gel vic in di xy ra. c. 0,5X TBE vo b m di ca thit b, lng m cn phi cho bn gel nm trong m. d. m vo b trn ca thit b, m bo gel nm di lp m. Kim tra xem m c b chy ra hay khng. e. Chy th 20-30 pht ti hiu in th 2000V (phi cn thn v rt nguy him), sau nhit ca bn gel nn t ti 50oC. 10, X l nhit vi cc mu ADN in di ti 85oC trong 5 ht v trn u, trn cho n khi ln mu. 11, Tt ngun in vo thit b in di (nh ngt ngun in vo thit b trong bt k trng hp no khi thao tc vi thit b). 12, Trong khi x l nhit vi cc mu th ra b mt vi m trong b in di, dng pipet hay syringer 60 ml. Mc ch ca thao tc ny l loi lp urea trn b mt gel. 13, nh du v tr nh ca mt gel trn bn knh trc ca gel. 14, t lc rng c mp ln mt gel, cc rng c t trn mt gel v ngp su vo mt gel khong 1 mm. Chc chn l vng khng gian gia cc rng c gii hn gia cc rng v gel. Nh vy phn lc rng c mp tr thnh cc cnh bn ca ging, b mt gel l y ca ging tra mu (hnh 1.10).

Hnh 1.10. V tr t lc sau khi gel. Phi m bo chc chn lm sch b mt gel trc khi t lc rng c mp ln trn gel. Nu khng thc hin vic ny s khng c c phn kt qu pha trn ca gel. Nu quyn thc hin bc ny c th ra sch tng ging tra mu nhng vic ny s kh khn hn. Nu ln nhiu mu th trc cn phi phi lm sch ging v urea s khuych tn v tch t ti b mt gel ti thi im ln mu. 15, Vic x l nhit 85oC i vi cc mu in di nhm lm bin tnh ADN, ch ln khong 2-4 ml mu cho mi ging (ln mu theo th t ACGT), c th dng bt vit ln knh nhm trnh nhm ln. 16, Qu trnh in di vi hiu in th 2000V cho n khi mu BPB (Bromophenolblue) di chuyn n tn cng bng gel (1-2 gi). Nu chy gel c gradient mui s cho php c c nhiu base hn trn mt gel do vic lm ngn li khong cch gia cc bng ADN phn cui bn gel. Nn dng in di t 45 pht 1 gi. Tt ngun in, b sung 1/2 th tch 3M NaOAc vo b m y, trn u. Li tip tc cng vic chy gel cho n khi BPB di chuyn n cui bn gel. Ch s c mt ca mui nng cao s dn n nhit tng, do cn phi kim sot nhit ca qu trnh chy gel nhm trnh v knh v hng gel. Chun b gel cho pht hin bng nh phng x. 17, Ly bn gel ra khi thit b, ch b m y cha cht phng x, cc nucleotid d s i ra khi bn gel. 18, b b m trn. 19, t bn gel trn bn th nghim sao cho tm knh ngn ln trn. Ph ln mt lp SaranWrap, dng lnh t ln trn khong 2 pht cho gel co li, dng thanh nha tch cc lp knh ra, gi nguyn khng cho tm knh trn tip xc li vi gel v nh vy gel s dnh v b v. Lc ny gel s ch dnh vo tm knh di khng c silicon c ph khi gel. 20, t tm giy Whatman ln trn gel v vut u nh nhng, lc gel s dnh vo giy v c th ly ra d dng (Ch trong trng hp s dng cht phng x l S35 th nn c nh mu theo cch di y). 21, Sau khi ly gel ra hi tm knh y th bc gel vi SaranWrap, ch trnh khng kh i vo gia lp SaranWrap v gel, iu ny s cn tr nh phng x trn phim. 22, C th a vo thit b sy gel.
23, Sy gel trong khong 45 pht ti 80oC. 24, t phim X-ray theo ng ch dn vo gel trong hp cassette (c phim ch c mt mt th mi thao tc phi ng c nh phng x). Thi gian nhn c bc x l 12-14 gi i vi P32 v 24-48 gi i vi S35.

25, c kt qu. C nh gel: Mc ch ca bc ny l loi bt urea khi gel v s c mt ca urea s hn ch s phn cch gia cc ADN, cc bc c thc hin nh sau: + t bn knh c gel vo khay chm trong hn dch 15% ethanol v 10% acetic acid. + C nh trong 10-15 pht urea khuych tn khi gel. + Dng giy lc thm ht dch c nh khi gel. + Tip tc lm theo cc bc nh quy trnh hin nh phng x.

Ha cht v cch chun b: Phng php: Gii trnh t ADN si kp s dng KIT bin tnh nhanh vi sequenase. Phng php ny thch ng vi vic gii trnh t cc plasmid si i. Bt u bng vic gy bin tnh trong kim (NaOH) hoc glycerol, sau kt ta ADN v bt cp vi ADN mi dng gii trnh t. Phng php gii trnh t ADN vi si i cn lng ADN mi cao gp 10-20 ln so vi si n. Vic gii trnh t gm hai phn ng: phn ng nh du v phn ng dng ti im cui. Trong phn ng nh du cc si ADN b sung c gn kt vi cht nh du phng x trong vic ko di chui. Sau phn ng ko di chui ADN b dng mt cch ngu nhin do nng thp ca nucleotid trong dch phn ng (0.08 mM). Bc tip theo l kt thc chi vi dideoxynucleotid khi c nng cao ca deoxyribonucleotid (33,3 mM). Cc trnh t tip theo ca si khun s c xc nh vi nng cao ca deoxynucleotid trong khi phn ng nh du xy ra trc vic b sung dideoxynucleotid vo hn hp phn ng. Quy trnh sau y c rt gn, c nhiu phn tin ch chi tit hn v thay i trong cc ti liu hng dn. iu quan trng c kt qu tt trong vic gii trnh t ADN l cht lng cao ca ADN plasmid. Trong cc khu chun b vic xut hin cc phn t t gy s dn n kt qu khng r, kh xc nh cc bng. Cc plasmid si n phi c lm bin tnh cho m xon trc khi gii trnh t. Bin tnh cc plasmid xon do lin kt ha tr khng phi lc no cng t c bng x l nhit ti nhit bt cp mi hay trong m phn ng, bi l nhit m xon thng l 105ng l 105oC. C hai phng php kh nhanh v tin li: Mt phng php xut pht t thc t l glycerol hoc ethylen glycerol c tc dng lm gim nhit m xon, cn phng php th 2 l m xon bng mi trng kim. C hai phng php u thc hin n gin cho hu ht cc loi ADN khun. i vi hai phng php bin tnh ADN, nn dng lng ADN khun

nng 0.5 pmol (05- 0.3 mg) v lng primer 2-5 pmpol. Nu s dng th tch ADN nh hn th phi pha n nng thch hp bng nc. Kt qu tt nht thu c l theo t l phn t mi : si khun l 4 : 1. T l ny nn c duy tr khi tin hnh vi lng ADN khun khc nhau. Trong mi trng hp khi m s lng thnh phn no qu t th u dn n bng khng r cui bn gel. Lng ADN khun cn theo phng php ny t hn so vi s dng sequenase KIT 2.0 do khng b mt ADN trong bc kt ta vi ethanol. Tc nhn gy bin tnh ADN khun trong KIT ny l m 40:50 glycerol v ethylen glycerol. Vic b sung m ny vo dung dch ADN theo t l ch dn; 40% glycerol s lm gim nhit bin tnh ADN xung khong 20oC. Ch khi dng m glycerol s gy nn vic di chuyn khng u trn gel in di ti vng c khong cch 300-400 k t on ADN mi. Nn s dng m chy gel hn ch tc dng ca glycerol (thay taurin cho m chy gel TBE) s khc phc c iu ny. ADN plasmid s bin tnh ti bt k nhit no pH kim (pH 13). iu quan trng i vi phng php ny l vic trung ha bc bin tnh kim bng HCL phi c thc hin mt cch chnh xc. Nh vy nn dng mt loi pipet cho vic b sung kim v trung ha bng acid HCL sau . Ni chung dung dch NaOH v HCL c chun b c nng 1M, trong trng hp dng ha cht ring phi m bo nng ch dao ng trong khong 0,95- 1.05M. Bc gy bin tnh kim v kt ta vi ethanol cng c th c thc hin vi cc ha cht trong KIT nhng i khi cng khng t c kt qu mong mun v cn phi ci tin. Trong trng hp gii trnh t vi ADN si n, khng phi thc hin bc bin tnh ADN, hn dch c chun b n gin gm: ADN plasmid, 05 pmol primer, 2 ml m phn ng v b sung nc cho 15 ml. Phn ng tt s c c trnh t di khong 400 bps. Cch tin hnh c th: 1. Bin tnh ADN (theo hai cch trnh by trn). A. Bin tnh vi glycerol: ADN: m bin tnh plasmid: Primer: Nc ct dn n: 0.5 0.3 mg (th tch ti a 7 ml). 5 ml. 1 ml. 13 ml.

Trn u, trong 5 pht 90 100 oC, lm lnh nhanh trn . B sung thm m phn ng: 2 ml t c tng th tch l 15 ml. B. Bin tnh vi kim (khng cn kt ta vi ethanol).

Thnh phn phn ng gm: ADN: NaOH 1M: Primer: 0.5 - 3mg (th tch ti a l 8ml) 2 ml. 1 ml.

Nc ct dn n: 11 ml. Trn u, gi trong 10 pht ti 37oC, lm lnh nhanh trn . iu quan trng trong phng php ny l bc trung ha phi c thc hin chnh xc vi HCL. Sau b sung HCL 1M : 2 ml. m phn ng plasmid: Tng th tch l: 2. Bt cp mi vo si khun: Gi hn dch ADN v mi 37oC trong 10 pht, sau gi trn lnh (mu ch nn dng trong vng 4 gi). 3. Chun b tube cha hn dch kt thc chui: Trong khi thc hin bc bt cp si khun v si mi, tin thnh ht 2,5 ml hn dch kt thc chui cho tng loi (ddA, ddC, ddG, ddT) cho vo tng tube ring bit. y np, trn dng cho cc bc 5 v 7. 4. Pha long dch nh du: Pha long dch nh du 5 ln vi nc v gi trn . Khng nn pha long dch nh du khi mun c on xa mi. Dch nh du: 2 ml Nc: Tng s: 8 ml 10 ml. 2 ml. 15 ml.

5. Lm m cc mu chun b trong bc 3: Bn mu c chun b trong bc 3 ln lt l ACGT c 37oC trong 1 pht. 6. Phn ng nh du (k t bc ny th mi u cn v pipt s thc hin vi ha cht phng x, do cn phi c cc bin php bo v v eo gng tay). B sung ln lt cc thnh phn sau vo 15 ml hn dch bt cp c chun b trong bc 1. DTT 0.1M : 1 ml

Dch nh du (bc 4): Ha cht phng x: S35 (P33, P32): Enzym T7 sequenase cho plasmid: Tng th tch :

2 ml 0.5 ml 2 ml 20.5 ml

(Th tch ny s c chia ra 4 phn trong bc 7) Trn u (trnh bt), hn dch ti nhit phng 2 -5 pht (i vi plasmid lm bin tnh trong glycerol th 5-10 pht ti nhit phng hoc 5 pht ti 37oC). Thng b sung hn dch T7 sequenase cho plasmid cui cng, sau khi cc thnh phn khc c b sung. Khng bao gi b sung hn dch T7 sequenase cho plasmid vo hn dch nh du, DTT hay hn dch khc khng phi l m. Ch : Nu trong trng hp cht phng x S35 qu lu th c th tng lng ln 2 ml cho mi phn ng, sau gim lng nc tng ng. 7. Phn ng kt thc chui: a. Ly 4,5 ml hn dch thu c t bc 6 cho vo thnh trn ca 4 tube kt thc chui c lm m bc 5 (A, C, G, T). y np li v li tm nhanh trn u, tip tc trong 5 pht ti 37oC (c th ko di ti 30 pht). Nu thy cn thit th c th li tm nhanh thu ly hn dch trc khi tin hnh phn ng nh du. b. Ti thi im cui ca 5 pht, b sung 4 ml hn dch dng phn ng vo thnh trn ca cc tube v ng np nhanh. Nh vy c th dng phn ng ti thi gian thch hp bng cch li tm dch dng phn ng i xung hn dch phn ng. c. Ngay lp tc li tm dch dng phn ng i xung hn dch phn ng, sau bc ny phn ng nh du hon tt v mu c th c gi 4 oC s dng trong ngy hm sau. d. X l nhit 75oC cho cc mu khong 2-3 pht ngay trc khi tra mu ln gel, vi cc mu cha glycerol th vic ln mu d dng vi u cn vng. e. Ln khong 2-3 ml mu cho mi ging trn gel gii trnh t. Nn a mt lng ng mu trn cc ging. Cch tt nht ln mu khng b bt l ht 3 ml mu nhng khi tra ch dng 2 ml l . Ha cht: Hn dch kt thc chui:

Tube dATP

A 80M

C 80M

G 80M

T 80M

dCTP 7-deaseGTP dTTP ddATP ddCTP ddGTP ddTTP NaCl

80M 80M 80M 8M

80M 80M 80M 8M

80M 80M 80M

80M 80M 80M

8M 8M 50 mM 50 mM 50 mM 50 mM

Dch dng phn ng: 95% Formamide 20mM EDTA (Na2), pH 8.0 0.05% Bromopheno Blue (BPB). 0.5% Xylen CyanolFF (XC). Ch , nu bn gp trc trc vi vic gii trnh t ADN th cc plasmid nn c ti tinh sch vi Genee-Clean hoc sc k trao i ion.

Hnh 1.11. Tra mu trn thit b chy gel. Cc phn di y cp n mt s vn ny sinh v cch gii quyt khi tin hnh gii trnh t ADN. - Mun c c trnh t ADN di hn: Yu cu u tin l phi c c on gene n mc c th trn gel. Thng thng th kch thc c cho mi phn ng trn gel l

250-300 nucleotid. Vi thi gian chy gel lu khong 8 gi th c th c c kch thc gene di hn 400 nucleotid. Nu s dng gel gradient s khng ph hp cho mc ch ny v khong cch gia cc bng ADN s hp ti mc khng th c c. Nng gel acrylamid dao ng 4-6% l thch hp cho kt qu tt. C mt gii php na l c th s dng gel vi gradient mui nhng vn gi nguyn thi gian chy gel (xem phng php 2, bc 16). - S dn cc bng: Nguyn nhn chung khng th hon tt vic gii trnh t ADN l cc bng khng r do cu trc bc hai trong sn phm ca phn ng ko di chui, iu ny thng c m t l s dn p cc bng do cc bng b thu hp mt cch bt thng trn nh fim ca kt qu gii trnh t ADN. Cc khong cch hon ton nh nhau khi m khong 3 lin kt G-C b sung km vi 2-5 base c th dn n kt qu khng th c c on gene chim mt khong nh trn bn gel. Khong trng bt thng l do nh hng ca chui vi khong cch ngn ca cu trc bc 2 s c cng tc di chuyn nh l si thng c chiu di ngn, ti v tr bn fim ngoi vng dn bng th khong cch cc bng s rng khc thng do s km bn ca cu trc bc 2 lm cho chiu di ca chui tng ln v s di ng ca chui tr nn nh bnh thng. Vic gii trnh t qua vng gene dn nn c th c khc phc bng cch thc hin vic c trnh t trn c hai si. S bt thng v vic di chuyn ny sinh cc chui c chiu di khng cho vic bt cp. iu ny c ngha vng gene c cc bng khng r rng s c khc phc bng cch s dng dITP thay cho dGTP trong phn ng gii trnh t ADN. L do l base I ch c 2 lin kt vi C thay v 3 lin kt trong trng hp vi G, kt qu l ADN s d dui hn. K thut ny thng c dng, do dITP c trong KIT sequenase. Mt cch khc c th lm km bn vng base dn nn do cn cp i l thc hin chy gel trong iu kin bin tnh (v d s dng formamide trong nc dng gel). Trong trng hp ny khi thay th 25% formamide cho nc s t c kt qu tt nht. Gel c formamide thng c dng khi vng dn nn kh gn mi, nn x l formamide qua gel trao i ion ngay trc khi chun b gel. Trong cc trng hp m cc cu trc cp i phc tp hn khi m vng dn nn li nm ngay ti v tr tng ng trn si i din. Khi cch gii quyt s tr nn kh khn v vi cc k thut hin c thng a li cc kt qu khc nhau. - Cng cc bng thiu s ng nht: Mt vn khc trong phn ng gii trnh t ADN l cng cc bng khng ng nht. iu ny thng xut hin trong phng php gii trnh t vi thit b t ng da vo phng php pht hin bc x hunh

quang. S thiu ng nht l do s c mt ca sequenase cn Mg++ gn dideoxy kt thc chui khc nhau ti cc v tr khc nhau (vn ny c bn lun k trong ti liu USB sequenase). b. K thut gii trnh t ADN trn thit b gii trnh t ADN t ng:

Hnh 1.12. Thit b gii trnh t ADN t ngg (3100-Avant Genetic Analyzer) Trong phn ny chng ti cp phn ng gii trnh t ADN trn 2 loi thit b ch yu hin ang c dng ph bin l: Beckman Coulter v ABI 3100-Avant Chun b mu gii trnh t ADN: 1. Thnh phn phn ng (cho 1 phn ng 20 ml): 80-100 ng 300ng) 3.2 pmol 20 l

- Terminator Ready Reaction Mix*: 8 l - Template: ( vi plasmid l: - Primer : - Nc ct v trnh:

Chun b Teminator Ready Reaction Mix cho 4 phn ng:

15 l Bigdye Sequencing Buffer 3.1 (5X) + 15 l nc = 30 l (2.5X) 20 l m 2.5X + 20 l Ready Reaction Premix= 40 l Terminator Ready Reaction Mix (dng 8 l) cho mt phn ng. 2. Tin hnh phn ng: Phn ng c thc hin trong 200 ml tube hay trong microplate. Cycling c thc hin trong my PCR theo chng trnh sau:
Denature 96oC: 96 C: 50 C: 60 C: Cycling:
o o o

1 pht 10 giy 5 giy 4 pht 25 cycles.

3. Kt ta sn phm PCR: - Cho vo 5l EDTA 125 mM + 60l E-OH 100%E-OH 100% - Trn u, 15 pht nhit phng. Ly tm 15 000 v/pht - Ra li bng 60l E-OH 70% v li ly tm (4oC) - Lm kh mu nhit phng (Speed Vac). - Khi mu kh, thm 10l Hi-Di formamide, trn u sau 96oC trong 2 pht.

- trn trc khi cho vo seq. 4. a mu vo my c trnh t.

Phng

th

nghim

"o"

http://web.mit.edu/7.02/virtual_lab/PBC/PBC5virtuallab.html
Formazan gm MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-dihphenyl-2H-tetrazolium bromide) v nc ha tan XTT (23-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium5-carboxanilide).

http://vietsciences.free.fr v http://vietsciences.org Dng Vn Hp v Nguyn Ln Dng