TNG LIN ON LAO NG VIT NAM TRNG I HC TN C THNG
THUYT TRNH: K THUT DI TRUYN
GING VIN HNG DN: TS. TRN TH DUNG SINH VIN THC HIN: TRN MINH TN NGUYN VN NGC MSSV: 080568H MSSV: 080550H
NI DUNG BO COI. GII THIU: II. TI SAO PHI XC NH TRNH T DNA: III. LCH S PHT TRIN IV. CC PHNG PHP XC NH TRNH T DNA: IV.1 Phng php gii trnh t gen theo phng php ha hc (Alan Maxam v Walter Gilbert): IV.2 Phng php dideoxy ca F.Sanger (1977): a.Gii thiu v phng php dideoxy ca F.Sanger (1977): b. Vt liu: c. Phng php: c.1).To ra defective nucleotide: c.2). Xc nh trnh t DNA template: IV.3 Gii m trnh t bng h thng t ng: V. NG DUNG CA VIC XC NH TRNH T DNA VI. TI LIU THAM KHO I. GII THIU:
- Trong sinh hc, xc nh trnh t DNA l cng vic, i hi cc phng php thc nghim, xc nh trnh t ca DNA. - Hin nay, trong lnh vc sinh hc ni chung v sinh hc phn t ni ring th vic gii trnh t nucleotide ca mt on gen hay ca c b gen ca mt loi sinh vt l mt k thut ht sc quan trng. - Trong di truyn hc v ho sinh, xc nh trnh t c ngha l xc nh cu trc chnh (hay trnh t chnh) ca mt biopolymer cha c phn loi. Xc nh trnh t cho kt qu l m t tuyn tnh mt cch hnh nh hay cn gi l "chui", tm tt sc tch cu trc mc nguyn t ca phn t va c xc nh trnh t. - Trong thut ng di truyn hc, xc nh trnh t DNA l qu trnh xc nh trt t nucleotide ca mt on DNA. Hin nay, hu ht mi xc nh trnh t DNA u c thc thi dng phng php phn tch trnh t (chain termination method) , c pht trin bi Frederick Sanger. K thut ny dng phn tch trnh t c th (sequence-specific termination) ca mt phn ng tng hp DNA trong ng nghim (in vitro) dng cht nn nucleotide c chnh sa. II. TI SAO PHI XC NH TRNH T DNA? - Trnh t ca DNA m ha cc thng tin cn thit cho cc c th sng c th tn ti v ti sn sinh. Vic xc nh trnh t v th rt hu ch vi cc nghin cu 'thun ty' l gii ti sao v bng cch no m cc c th tn ti, cng nh cc ch mang tnh ng dng. - V bn cht quan trng ca DNA i vi cc sinh vt sng, hiu bit v trnh t DNA c th tr nn hu ch vi cc nghin cu sinh hc v ng dng. V d, trong y khoa n c th c dng xc nh, chn on v pht trin cc phng php iu tr cho cc bnh v di truyn hc. Tng t, cc nghin cu vo pathogens c th gip iu tr cc bnh ly nhim (contagious diseases). K thut sinh hc (biotechnology) l mt ngnh ang pht trin, vi tim nng p dng cho cc sn phm v dch v hu ch.
III. LCH S PHT TRIN. 1870 Mischer : Pht minh DNA.. 1940 Avery: DNA l cht liu di truyn .
1953 Watson & Crick: Cu trc xon kp ca DNA. 1965 Holley: Trnh t ca tRNA mm men. 1977 Wu: Trnh t ca u dnh DNA trc khun . 1977 Sanger: Phng php xc nh tnh t dideoxy. 1977 Maxam & Gilbert: Xc nh trnh t DNA bng phng php ha hc. 1980 Messing: Trng dng trong trc khun M13. 1986 Hood et al: T ng ha mt phn. 1986 Hood et al: T ng ho mt phn. 1990 Phn tch trnh t bng chu trnh nhit (PCR); cc enzyme c hon thin c trnh t; cc h thng d hunh quang c hon thin (c qua ng mao dn) 2000 2003 Gii m thnh cng b gien ngi . 2002 T ng ho phng th nghim. IV. CC PHNG PHP XC NH TRNH T DNA: IV.1 Phng php gii trnh t gen theo phng php ha hc (Alan Maxam v Walter Gilbert):
- Phng php ca Maxam v Gilbert (1977) ct t si i DNA ti cc v tr c bit x mt x l bng P32 ha hc lm cc phn bin i ng u mt hay hai ho hc. 5 `. base. - Cc on nucleotide ging nhau (v d, di 16 N) c nh du ng v phng - Cc on mang du phng x ny c chia thnh 4 nhm, mi nhm chu
- C th l x l methyl ha (bng dimethyl sulfate) lm mch t G. - pH = 2 lm t A hoc G. - Hydrazine lm t C v T. - Hydrazine nng mui cao lm t C. - X l trung bnh mi on nucleotide ch hng mt base.
- Sau khi x l, mi hm cha mt s on nucleotide c chiu di khc nhau. Bn nhm c a ln 4 bn gel acrylamide ring phia cc m, cc on ngn hn s di chuyn xa hn v pha cc dng. - Da vo kt qu in di c th xp cc on DNA theo th t t ngn n di c im mc chun l u 5` mang du phng x P32 c pht hin nh phng php phng x t ghi (autoradiography) biu hin vt en trn giy in nh. - Tng hp kt qu ca 4 nhm trn 4 bn in di phn nh y trnh t ca 16 nucleotide ca on DNA k t u 5 `.
www.cbs.dtu.dk=>Hin nay ngi ta khng s dng rng ri phng php ho hc do c tnh ca ho cht.
IV.2 Phng php dideoxy ca F.Sanger (1977):
a.Gii thiu v phng php dideoxy ca F.Sanger (1977): - n thp nin 1970, Sanger a ra phng php xc nh chnh xc trnh t chui nucleotides trong mt gene. Phng php ny da trn kt qu ca Arthur Kornberg v qu trnh t nhn i ca DNA (DNA replication) - mt chui DNA mi c tng hp da vo thng tin t mt chui DNA mu) nh vo enzyme DNA polymerase III kt ni cc nucleotide ring r. - V d DNA template l mt poly-thymine DNA, th DNA strand mi to thnh s cha ton Adenine (theo nguyn tc bt cp (base-pair): A-T, G-C).
Gii thch hnh trn: Trong qu trnh hnh thnh chui DNA mi, phosphate u C-5 ca deoxynucleotide triphosphate (dNTP), trong trng hp ny l dATP, t do i vo s gn vi u C-3 cui chui mi. Tip n, lin kt ester gia hai nhm hydroxyl 2 chui (strand) s c to ra. Theo cch ny, chui nucleotides mi s c ni di da trn thng tin ca chui nucleotides lm mu (template (ddNTP). - ddNTP so vi dNTP th thiu mt nhm hydroxyl(-OH) u C-3 do cc dNTPs t do s khng gn c vo u C-3 ca n thng qua lin kt phosphodiester. strand). - Phng php Sanger mu cht vic s dng dideoxynucleotides triphosphate
=>Tm li, trong qu trnh tng hp nn chui DNA mi, enzyme DNA polymerase xc tc gn cc nucleotides vo si n DNA theo nguyn tc b sung tng t nh qu trnh sao chp DNA. Nu nh mt ddNTPs no gn vo chui ang c tng hp th qu trnh tng hp s b gin on, do phng php ny cn c gi l phng php gin on chui( chain determination method ).
b. Vt liu: - Primer( 20 nucleotides). - DNA polymerase
- DNA template - dNTPs v ddNTPs - Vt liu nh du - H thng in di c. Phng php: c.1).To ra defective nucleotide: - Da vo vic s dng cc dideoxynucleotide(ddNTP) kt thc chui(chain terminating dideoxynucleotide) th Sanger thit lp ra 4 phn ng din ra ti 4 l c th u nghim ging ring nhau cho bit c nh 4 sau: l). + Mi l phn ng gm: mt DNA template, mt primer ,DNA polymerase III (tt + Mt hn hp cc dNTPs trong c mt loi dNTP c nh nhn phng x hoc c nhum mu hunh quang ty theo tng l; v d l 1 th c dATP c nh nhn phng x, l 2 th dCTP, l 3 dTTP v l 4 l dGTP.
+ Sau , mi loi didNTP A, T, C, hoc G c thm vo mi l th nghim c lp trn (v d: l 1 th c thm didNTP Adenine, l 2 th c thm
didNTP Cytosine). Vi cc nguyn liu nu trn, qu trnh tng hp ra nucleotide strand mi s c din ra trn mi l. N s dng li khi c mt didNTP c gn vo. Do , chiu di ca DNA strand mi l ngu nhin v di ngn khc nhau mi l phn ng. Nng (concentration) ca didNTP c thm vo thp hn rt nhiu so vi cc dNTP c sn > gip m bo l DNA strand to ra di.
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+ Tng nhit trong l ln khong 100oC khi chui DNA s tch ra thnh 2 si n, mt trong 2 si s lm si khun(DNA template). Sau nhit trong l s c h thp xung khong 600 ,lc ny primer s li gn vo DNA template tng ng vi n v DNA polymerase III s xc tc cho qu trnh sao chp v qu trnh tng hp bt u. + Qu trnh tng hp to ra chui DNA mi s din ra cho ti khi c mt ddNTP(ddATP) ti b sung vo chui mi th phn ng lc ny cng dng li.
+ Ty thuc vo thi im ddNTP-Adenine gn vo m on DNA (DNA fragments) thu c s c chiu di ngn khc nhau.
+ Kt qu cng tng t vi 3 l cn li: mi l s c cc DNA fragments mi vi nucleotides kt thc chui tng ng l ddCTP, ddTTP v ddGTP.
c.2 Xc nh trnh t DNA template: - Trnh t ca chui DNA template c th xc nh gin tip thng qua trnh t ca chui DNA mi to thnh, da trn nguyn l b sung cho nhau. - Lc ny ta nung nng mi l tch cc DNA fragments mi ra khi DNA template. - DNA fragments s c a ring r vo mt mi trng gi l polyacrylamide gel in di.
- Mi fragment s c ngn cch nhau nh vo urea gip ngn cc DNA fragments hai l khc nhau khi gn kt vi nhau trong qu trnh in phn (electrophoresis). - Qu trnh electrophoresis s gip 4 bands di chuyn t cc (-) sang cc (+).
-Ngoi ra theo di qu trnh dch chuyn ca cc DNA fragments, mt cht nhum nhanh mu mu xanh cng c a vo. hn. - Qua qu trnh electrophoresis, cc DNA fragments ngn hn th dch chuyn - Cc DNA fragments c cng kch thc s dch chuyn v cng 1 v tr. - Kt thc qu trnh electrophoresis, gel matrix s c chiu ln tia X v chp hnh li gip nhn dng cc v tr ca cc dNTPs c nh nhn.
- Trnh t ca DNA template s c xc nh chnh xc thng qua vic c trnh t t nucleotide di cc bands. ln. - c t cc bands c kch thc nh nht n cc bands ln hn lin k v c
IV.3 Gii m trnh t bng h thng t ng: - Vic c trnh t ca DNA theo cch th cng tng i chm, kh khn v d sai st. - Hin nay k thut c trnh t DNA t ng (automated DNA sequenced) ra i v c s dng rng ri hn thay th cho vic c th cng.
MY GII M TRNH T NG DNA
- Cc h thng ny s dng h thng pht hin mu hunh quang (fluorescent), ha cht pht sng (chemiluminescent) hoc h thng o mu (colorimetric). - Vic s dng cc primer hoc dideoxynucleotide c gn hunh quang gip cho phng php ny tr nn ph bin. - V nguyn tc th mt mch khun DNA c c trnh t bng cch s dng phng php tng t bc ko di primer trong k thut PCR. - Mi mt trong 4 dideoxynucleotide khc nhau c gn vi mt loi mu hunh quang cho php pht ra mt ph nh sng ring. - Cc on DNA c gn hunh quang sau phn ng tng hp s c in di trn gel polyacrylamide rt mng ri sau c kch thch bng mt chm tia laser . - nh sng pht ra c ghi nhn qua mt camera k thut s chuyn thnh cc tn hiu in t v chuyn thnh nh. - nh ny c phn tch chuyn thnh dng th.
- Trong mi mt loi nucleotide khc nhau c m t bng mt nh mu khc nhau, trnh t ca cc nh mu ny trn th cho php c trnh t ca on DNA.
- Mt hng c trnh t t ng khc l cc mu DNA c in di trong cc ng thu tinh rt nh c gi l cc ng mao dn (capillary). - V nhng ng ny rt mng v lng nhit ta ra trong qu trnh in di rt t nn vic c trnh t din ra rt nhanh, k thut ny cho php c trnh t ca 600 bp ca 96 mu ch trong vng 15 pht.
My phn tch trnh t gen t ng-capillary
- Ngoi ra mt k thut mi l s dng sc k khi ph (mass spectroscopy), y l mt k thut c phn gii cao trc y dng phn tch protein. S pht trin ca k thut MALDI-TOF (matrixassisted laser desorption/ionization time-of-flight) cho php phn tch hng trm mu DNA trong vi pht. Hin nay y l k thut thu ht nhiu s ch .
MY SC K KHI PH
Trnh t DNA khi chy trn my tnh
=>Tm li: - phng php gii trnh t bng h thng t ng cng c da trn nguyn tc lm gin on chui ang tng hp ti mt im bt k no trn DNA khun bng mt loi nucleotide ci tin thnh dng ddNTP. - Vi k thut ny th primer hay cc ddNTP c nh du phng x v cc sn phm DNA c khuch i tng t nh trong k thut PCR. Ch cn mt phn ng duy nht v sn phm to ra l mt dy cc phn t DNA m phn t ny di hn phn t kia mt nucleotide.
V. NG DUNG CA VIC XC NH TRNH T DNA. - Nghin cu s a dng v tin ho ca sinh vt. - Gii m trnh t b gien ngi v vi sinh vt khc .
- ng dng trong nghin cu v iu tr mt s bnh di truyn. VI. TI LIU THAM KHO. - Phm th anh th : Laboratoire Ecophysiologie Moleculaire, Universite Paris 12. - phng php xc nh trnh t DNA ca PGS.TS. NNG VN HI ( Vin cng ngh sinh hc ). - DNA sequencing: Dideoxy method ca ThS. PHM TH B T. - Ti liu trn mng , sch bo, din n sinh hc. - www.technimexvn.com . - www.azcobiotech.com. - www.scq.ubc.ca.- http://en.wikipedia.org/wiki/DNA_sequencing
