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NGUYN VN DUY NHM : 8 LP: 52CNSH

1. NGUYN XUN V

2. BI TH KHNH NG
3. INH TH NGC YN 4. NGUYN MAI SAN NY

TNG QUAN

NGUYN TC XC NH TRNH T DNA

CC PHNG PHP THC HIN

NG DNG CA PHNG PHP

A. Khi nim: Trong thut ng di truyn hc, xc nh trnh t DNA l qu trnh xc nh trt t nucleotide ca mt on 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

Gii m trnh t cc nucleotit c th xem l vic nh du mu d trit nht ca mt h gen vi tnh chn lc cao.

B. Mc ch: Xc nh chnh xc trt t sp xp ca cc cp trng mt on DNA

Phn tch trnh t ca cc n v mang thng tin di truyn. L gii ti sao v bng cch no m cc c th tn ti
Hiu bit v trnh t DNA c th tr nn hu ch vi cc nghin cu sinh hc v ng dng So snh gia tng ng gen gia cc loi v xc nh t bin

 L k thut xc nh tt c nhng hp phn do nucleotide hnh thnh nn phn t DNA Cc cng trnh u tin v DNA sequencing c cng b bi Maxam va Gilbert (1977), Sanger v cng s (1977)

Cu trc ca chui DNA l mt chui xon kp, hai mch n lin kt vi nhau bi lin kt hidro. Trn mi dy c mt chui cc base, tp hp ca bn nucleotide : A, T, C, G
K thut DNA sequencing s dng k thut in di vi gel loi polyacrylamide c phn gii cao.

PHNG PHP HA HC

3 TYPE

PHNG PHP ENZYME

PYROSEQUENCING

 Da vo s thy gii c trng phn t DNA cn xc nh trnh t bng phng php ha hc Nguyn tc ca phng php ho hc ca Allan Maxam v Walter Gilbert l s dng phng php nh du phng x (P32), x l ho hc ph hu 1 nucleotit to ra hng lot cc on ADN c kch thc khc nhau.

STEPS
Step 1
DNA si kp bin tnh thnh dng si n

Step 2
nh du phng x (P32) u 5'

Step 3
S dng ho cht ph hu mt loi nucleotit nht nh

Step 4
in di trn gel poliacrylamid

Ha cht
Dimethyl sulfate pH 8 Piperidine formate pH 2 Hydrazine Hydrazine + 1,5 M NaCl

Base b nh hng
G A+G A+ T C

Nhit cao (900C), 1,2 M NaOH

A >C

 Nguyn tc ca phng php ny l s dng deoxynucleotit khng c nhm OH v tr 3' do vy trong qu trnh ko di chui polinucleotit enzyme polimerase gn chng vo chui polinucleotit th qu trnh tng hp b dng li. Kt qu to ra cc on DNA c kch thc hn km nhau mt nucleotit, trn c s xc nh c trnh t nucleotit.

Frederick Sanger

STEPS
Step 1
DNA si kp bin tnh thnh dng si n

Step 2
Mi tip hp vi DNA si n.

Step 3
Phn ng tng hp chui polinucleotit

Step 4
in di trn gel poliacrylamid

 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.

 Mi loi ddNTP A, T, C, G c thm vo mi l th nghim c lp trn

 Tng nhit trong l ln khong 92oC n 96oC 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 600C ,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 ti b sung vo chui mi th phn ng lc ny cng dng li

 Ty thuc vo thi im ddNTP gn vo m on DNA (DNA fragments) thu c s c chiu di ngn khc nhau

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

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

IN DI

...V C KT QU

Vic xc nh trnh t Nucleotide n gin hn khi x dng cc h thng t ng Da trn phng php Sanger 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.

 Trong k thut ny, khng dng cc ng v phng x, thay vo l cc ha cht c kh nng pht hunh quang (fluochrome). Mi loi Dideoxynucleotide c nh du bng fluochrome c mu khc nhau. Tt c cc oligonucleotide cng chm dt ti mt loi Dideoxynucleotide s c cng mt mu.
in di kt qu c c nh h thng vi tnh.

 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.

 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/ionizatio n 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 .

 Sng to u tin bi Pal Nyrn v Mostafa Ronaghi ca Vin K thut Stockholm, Thy in nm 1996
Nguyn l gii trnh t bng vic tng hp da trn vic nhn bit cc pyrophosphate (PPi) c gii phng trong qu trnh gn nucleotide, to ra mt tn hiu nh sng, hiu qu hn k thut kt thc chui bng ddNTP.

K thut ny c tnh nhy cao hn hn so vi phng php gii trnh t truyn thng vi chnh xc l 99,9% vi cc on 200 bp v 99% vi cc on 400 bp

 u tin DNA c ct thnh cc on u bng (blunt end), v c gn cc oligonucleotide adaptor vo c hai u. Tng on ny sau gn vi mt ht thy tinh, v s c khuch i bng PCR trong cc git nh du-nc, to ra nhiu bn sao on DNA trn mi ht thy tinh. Sau cc ht thy tinh c gi trong cc tm picotiter trn mt c cht dng si, v gii trnh t.

 Si khun DNA n sau khi PCR, c lai vi mt primer gii trnh t v sau c vi cc enzyme DNA polymerase, ATP sulfurylase, luciferase v apyrase cng nh cc c cht adenosine 5 phosphosulfate (APS) v luciferin

 Khi dNTP u tin c bt cp b sung, n c gn vi si DNA khun nh DNA polymerase v km theo s gii phng mt PPi. PPi c bin i thnh ATP nh enzyme ATP sulfyrelase cng vi s tham gia ca APS, sau mt phn ng c xc tc bi licuferase s bin i luciferin thnh oxyluciferin - to ra nh snh nhn thy, c sng tng ng vi lng ATP to thnh. nh sng ny c nhn vit bi mt camera v phn tch bi mt chng trnh phn mm.

 u im ca k thut pyrosequencing l tnh linh hot cao khi kt cp primer, phn tch c nhiu t bin, d liu v thng tin trnh t tp hp c y .

H thng gii trnh t c 400-600 triu bp trong 10 gi v vy gi thnh thp hn khi gii trnh t lng ln DNA vi thi gian nht nh so vi khi s dng phng php Sanger mt nhiu ngy gii trnh t mt lng DNA tng ng

 Cc si khun em gii trnh t c th c chun b bng c hai phng php l: chun b si khun pha rn solid phase template preparation (streptavidin-coated magnetic bead, ht thy tinh t tnh c bc streptavidin) v chun b si khun mang enzyme enzymatic template preparation (apyrase v exonuclease).

Pht hin t bin Nghin cu qu trnh tin ho phn t Phc hi cc gen tn ti hng triu nm Chn ging vt nui, cy trng La chn cc cp cha m thun chng trong thi gian ngn Xc nh cc loi mi, cc loi c hu bng phng php di truyn phn t

Y hc Khoa hc hnh s.
Chun on chnh xc cc bnh nhim trng t vi khun, nm, virus Chun on sm cc bnh gy ra do ung th, cc bnh di truyn Xc nh huyt thng, truy tm du vt ti phm L k thut nn cho cc k thut khc nh: RAPD, SSR

Vit nam hin ti c hn 300.000 lit s v danh. Vic tr li tn cho cc lit v danh v a cc lit s v vi gia nh, ngi thn ca mnh l cng vic c ton x hi c bit quan tm

 Ty th l mt bo quan quan trng ca t bo, nm trong nguyn sinh cht ca t bo. Trnh t DNA ca gen ty th thng n nh qua mt thi gian di trong lch s tin ho v s khc bit trnh t ca gen ty th gia cc thnh vin c quan h di truyn theo dng m l rt him.

 ngi DNA ty th c 16569 cp baz (nucleotide) trong c cha tng cng 37 gen m ho cc sn phm, ngoi ra cn c vng khng m ho gi l vng kim sot hay vng D-Loop c cha nhiu c im cu trc DNA c trng cho c th v v vy n c s dng cho mc ch gim nh hnh s truy nguyn ngun gc c th v xc nh mi quan h huyt thng theo dng m.

S 1: Ph h xc nh di truyn theo dng m DNA ty th inheritance (maternal)

 Ly mt lng nh xng hi ct tch chit DNA ty th. Phng th nghim tch DNA ty th phi c mi trng rt sch v DNA ty th c kh nng nhim cao.

 Sau khi tch c DNA ty th t hi ct chng ta tin hnh nhn bi DNA vng D-loop bng my PCR thu c mt lng ln DNA ty th phc v cho vic gii trnh t.

 Gii trnh t DNA vng D-loop.

Sau khi gii c trnh t DNA ty th ca cc mu cn gim nh, cc trnh t ny c so snh vi nhau bng phn mm SeqScape2.5, s so snh ny u thng qua trnh t chun Anderson tm ra mi quan h huyt thng gia chng vi nhau

1. http://www.genomenewsnetwork.org/resources/timeline/1977_Gilbert.php

2. http://en.wikipedia.org/wiki/DNA_sequencing
3. http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Obenra der/sanger_method_page.htm

4. http://baigiang.violet.vn/present/same/entry_id/4791342
5. Phng php xc nh trnh t DNA ca PGS.TS. NNG VN HI ( Vin cng ngh sinh hc ). 6. Sinh hc phn t - H Hunh Thy Dng 7. Mt s ti liu trn internet..