Co So Di Truyen 1

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PHN I
C S DI TRUYN
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M U LC S PHT TRIN CA DI TRUYN HC V K THUT DI TRUYN
1.1- LNH VC NGHIN CU CA DI TRUYN V CNG NGH DI TRUYN Thut ng di truyn (geneties) xut pht t gc latinh l gentikos (ngun gc). Di truyn hc l mt b mn ca sinh hc, chuyn i su nghin cu hai c tnh c bn ca s sng l tnh di truyn v tnh bin d. Tnh di truyn biu hin s ging nhau ca cc tnh trng gia th h ny v th h khc. c tnh di truyn cho php th gii sinh vt bo ton ni ging. Tri qua nhiu th h ni tip nhau nhng nhng c tnh di truyn khng b mt i, th h con chu lun c nhng c im ging b m, ng b. Cc sinh gii sng trong iu kin mi trng lun c nhng bin ng nh s thay i thi tit, nhit mi trng, lng nc, lng thc n v s u tranh sinh tn gia cc loi. thch nghi vi iu kin sng, cc c th sng cng c nhng thay i, lm xut hin nhng tnh trng khc nhau gia cc th h, l s bin d. Bin d biu hin s sai khc ca th h con chu so vi th h b m ng thi s sai khc ca mt c th no so vi cc c th khc cng n. Di truyn hc thc s tr thnh mt b mn khoa hc c lp k t nhng nm 1900 - 35 nm sau ngy Mendel cng b cng trnh Cc th nghim lai thc vt. T n nay, cng vi s pht trin mnh m ca cc nghnh khoa hc khc nh vt l, ton hc, ha hc, di truyn hc v ang khm ph rt nhiu quy lut v s tn ti v lu truyn s sng v tr thnh mt mi nhn trong nghin cu sinh hc. Nhng thnh tu rc r ca di truyn hc em li nhng nhn thc mi v cu to v s vn hnh b my di truyn ca c th sng. Cng vi s pht trin mnh m ca di truyn hc, mt lnh vc nghin cu mi ca di truyn hc ra i, l k thut di truyn hay cn gi l cng ngh gen hoc cng ngh di truyn (genetic engineering).
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C th ni, k thut di truyn l mt tp hp ca nhiu k thut nh ha hc, sinh hc phn t, vi sinh vt hc,... m trong , vai tr hng u thuc v cc t duy v phng php ca di truyn. Cng ngh di truyn s dng cc phng php sinh hc phn t tch DNA t mt c th sng v sau ct, ni cc gen trn DNA. Bng cch nh vy, ngi ta c th loi b cc gen khng mong mun v a vo cc gen mi c hiu theo ch la chn. Cc thao tc ct, ni trn DNA c thc hin bn ngoi c th sng trong cc ng nghim (in vitro). Phn t DNA mi c to dng sau cc thao tc ct, ni c mt s c im khc vi phn t DNA ban u c tch ra t t bo sng, c gi l DNA ti t hp v k thut ny c gi l k thut ti t hp DNA. S ti t hp DNA c nh gi l thnh cng ch sau khi a c phn t DNA ti t hp vo trong t bo sng v chng biu hin cc hot tnh di truyn v th h con chu s mang phn t DNA ti t hp. C th ni: K thut di truyn l s thao tc b my di truyn ca mt c th sng bng cch thm vo hay loi bt gen c hiu. 1.2- GIAI ON DI TRUYN SAU MENDEL Pht minh ca Mendel t nn mng cho di truyn hc. Tuy nhin, thi im m Mendel cng b cng trnh nghin cu ca mnh, mt phn do cha hiu r c c ch phn bo, cc nh khoa hc cha th hiu v nh gi ng mc tm quan trng ca pht minh ny. Cui th k XIX, 5 nm sau ngy cng b cng trnh ca Mendel (1870), cc giai on ca qu trnh phn bo nguyn phn v sau , phn bo gim nhim (1890) c m t mt cch chi tit. Di knh hin vi, cc nh nghin cu quan st thy cc nhim sc th v s phn chia cc nhim sc th trong qu trnh phn bo. Nm 1902-1903, W.S Sutton, Th. Bovery v mt s nh khoa hc khc tin hnh cc nghin cu c lp, cng pht hin c s tng quan ng iu gia s biu hin ca nhim sc th trong phn bo vi s biu hin ca cc tnh trng theo Mendel. Thut ng gen do nh khoa hc an Mch W. Johansen nu ra nm 1909. Hc thuyt di truyn nhim sc th ra i: Cc gen c chng minh l nm trn nhim sc th, chim mt v tr xc nh, xp theo ng thng v chng chu s phn li nh nhim sc th.

Hc thuyt di truyn nhim sc th a di truyn hc ln mt bc pht trin mi. S pht trin ca di truyn nhim sc th gn lin vi nhm nghin cu do Morgan lnh o vi cc nh di truyn hc ni ting nh C. Bridges, A.H Sturtevant v G. Muller: - Vic pht hin ra s khc nhau gia cc c th c v ci 1 cp nhim sc th gi l nhim sc th gii tnh, l mt d kin quan trng xy dng nn hc thuyt di truyn nhim sc th. Cc gen nm trn nhim sc th gii tnh s c s di truyn khc hn so vi cc gen nm trn nhim sc th thng. Quy lut di truyn ca cc gen lin kt vi nhim sc th gii tnh c xc nh. - Hin tng trao i cho gia cc on nhim sc th tng ng trong phn bo gim nhim dn ti s sp xp li cc gen, t xut hin cc giao t dng mi khng ging ca cha m, gi l dng ti t hp (recombinant). Da vo tn s ti t hp n nh gia cc gen, ngi ta xy dng cc bn di truyn nhim sc th. Hc thuyt di truyn nhim sc th cng c thm cho hc thuyt v gen ca Mendel, nhng n c th hn, ng thi, n ch r gii hn ca quy lut phn li c lp trong hc thuyt ca Mendel. Sau chin tranh th gii ln th hai, di truyn hc phn t pht trin rt mnh m. Nm 1944, O. Avery, Mc. Leod v Mc. Carty chng minh rng: DNA chnh l cht di truyn. Nm 1953, m hnh cu trc DNA ca Wattson-Crick ra i, to mt bc ngot ln cho s pht trin ca di truyn hc v sinh hc. Nm 1961, M. Nirenberg v J. Matthei xc nh c b m di truyn u tin v sau , ton b cc b m di truyn cng c tm ra. 1.3- S RA I CA CNG NGH DI TRUYN K thut di truyn ra i vo nhng nm u ca thp nin 70 trong th k 20. Phn t DNA ti t hp u tin c nhm cc nh nghin cu M l Berg, Boyer v Cohen to ra nm 1972-1973 t ngun vt liu di truyn ban u l DNA ca virus SV40 gy ung th kh c ct, ghp vi DNA ca vi khun E. Coli. Phn t DNA mi c to ra trong ng nghim ny khng c a vo vi khun E. Coli nh d nh v l do an ton cho ngi v

ng vt, s rng loi vi khun mi mang gen ca virus gy nn ung th, nu thot ra ngoi s gy dch bnh m con ngi th cha c cch iu tr, tai hi l kh lng. Nm 1973-1974, nhm cc nh khoa hc ngi M do Cohen ng u vi Helinski, Boyer s dng plasmid lm ngun vt liu cho cc nghin cu ca h. Plasmid l nhng phn t DNA mch vng, xon kp, ngoi nhn. Plasmid xut hin kh ph bin vi khun, m ha cc gen biu hin tnh khng thuc, khng khng sinh vi sinh vt - plasmid pSR100 c phn lp v lm sch. Plasmid ny mang gen khng tetracycline c ct ti mt v tr bng enzyme ct hn ch EcoRI, v tr ct ny khng nm trong vng c cha cc gen c bn v ni mt on DNA ca plasmid R6-5d c cha gen khng kanamixin to thnh mt phn t lai gi l DNA ti t hp. Nh vy, phn t DNA ti t hp c cha hai gen biu hin tnh khng tetracycline v kanamixin. Phn t DNA c to dng trong ng nghim ny c a tr li vo t bo E. Coli. Kt qu l vi khun E. Coli mang plasmid c cha hai gen khng thuc c kh nng khng c tetracycline v kanamixin, ngha l, loi vi khun ny c th pht trin c trn mi trng chn lc c cha c tetracycline v kanamixin. Mt th nghim kim tra c b tr khng nh rng, loi vi khun mi ny mang phn t DNA ti t hp. Sau thnh cng rc r ca nghin cu trn, nhiu nh khoa hc tin hnh cc th nghim lp ghp gen v thu c nhiu kt qu ng dng c trong thc tin. T nhng nm 1990, s kt hp gia sinh hc, cng ngh di truyn v tin hc cho php rt ngn thi gian nghin cu v thu c nhiu kt qu hon ho hn. 1.4- NGHA KHOA HC V THC TIN CA CNG NGH DI TRUYN Cng ngh di truyn hin ang ng vai tr c s cho nhng nghin cu v ng dng ca cng ngh sinh hc, l mi nhn ca nghnh cng ngh sinh hc. V ngha khoa hc, c th ni rng, s ra i ca cng ngh di truyn v nhng thnh tu t c to nn mt cuc cch mng v nhn thc ca con ngi i vi th gii sinh hc. Ngy nay, con ngi hiu r hn v

cc c ch di truyn v ang tng bc iu khin chng to ra cc chng loi sinh vt c li cho con ngi. V nghi thc tin, cng ngh di truyn c ng dng trong nhiu lnh vc nghin cu v sn xut lin quan n nng nghip, cng ngh thc phm, y dc, bo v mi trng, ... Kh c th lit k ht tt c nhng kt qu nghin cu da trn c s ca cng ngh di truyn c ng dng v tc pht trin nhanh chng ca lnh vc cng ngh ny, xin gii thiu mt s v d c th nh sau: 1.4.1- Trong y dc Vn dng k thut di truyn, cc nh khoa hc c th chn on cc bnh do ri lon di truyn v tin ti iu tr bng cch thay gen bnh bng gen lnh hay a gen lnh vo c th b p cho gen bnh - y l mt hng ng dng kh thc hin nht. Trong nhng nm qua, lnh vc ng dng cng ngh di truyn mnh nht trong y t l nghnh sn xut thuc khng sinh, vc xin, khng th n dng v cc protein c hot tnh sinh hc. Hin nay, cc nghin cu nhm tm kim cc cht khng sinh mi tng mnh do hin tng vi sinh vt khng li tc dng ca khng sinh ngy cng nhiu hn. Phm vi ng dng ca khng th n dng trong ngnh y t ngy cng tng nh phn tch min dch, nh v cc khi u, pht hin mt s protein c lin quan n s hnh thnh khi u, xc nh s c mt ca cc loi vi khun khc nhau, ... gip cho cc bc s xc nh bnh mt cch nhanh chng v chnh xc. Khng th n dng l tp hp cc phn t khng th ng nht v mt cu trc v tnh cht. Khng th n dng c to ra bng cch cho lai t bo lympho trong h min dch ca ng vt hoc ca ngi vi t bo ung th. Mt s th lai c kh nng to ra khng th c hiu i vi khng nguyn. Chn cc th lai nhn ln v sn xut khng th n dng. Cc t bo lai c kh nng tng sinh vnh vin trong mi trng nui cy - tnh cht ny nhn c t t bo ung th. Nh cng ngh s dng DNA ti t hp m ngi ta c th sn xut mt s protein c hot tnh sinh hc dng cha bnh nh insulin cha bnh

tiu ng, interferon cha bnh ung th, cc hormon tng trng cho con ngi. Bn cht ca cng ngh ny l lm thay i b my di truyn ca t bo bng cch a gen m ha cho mt protein c hiu v bt n hot ng to ra mt lng ln loi protein m con ngi cn. 1.4.2- Trong nng nghip Vn chn ging c vai tr rt quan trng trong sn xut nng nghip, nhm nng cao sn lng v chng cc loi su, bnh cng nh cc iu kin t nhin bt li i vi cy trng v vt nui. K thut di truyn c s dng xc nh v tr ca cc gen m ha cho cc tnh trng mong mun, gip cho vic lai to, chn ging cng nh to ra cc sinh vt chuyn gen nhanh v hiu qu cao. Hin nay, nhiu ng vt v thc vt chuyn gen ra i, p ng nhu cu cho con ngi. Cy chuyn gen l cy c mang nhng gen c hiu m trc n khng c, do con ngi a vo n bng k thut di truyn. Cc gen c chuyn thng lin quan n cc tnh trng nh chu hn, chu mn, chng c su bnh, ... 1.4.3- Trong cng ngh thc phm Cng ngh ln men l mt lnh vc quan trng trong sn xut thc phm. Vic tuyn chn cc chng vi sinh vt c kh nng ln men tt, em li hiu qu cao l rt cn thit. Cc nghin cu s dng cng ngh di truyn phc v cho cng ngh ln men ch yu i vo hai hng chnh l: - Phn tch di truyn cc loi vi sinh vt s dng trong qu trnh ln men, xc nh cc gen m ha cho cc tnh trng mong mun nhm to ra nng sut v cht lng sn phm ln men. - To ra cc vi sinh vt chuyn gen phc v cho cc qui trnh ln men. V d trong sn xut ru, ngy nay ngi ta dng cc chng vi sinh vt c kh nng to ru cao v cho hng v tt. Phn ln cc chng c nghin cu, tuyn chn, lai to bng cng ngh di truyn. sn xut ru vang, trc y, ngi ta phi dng hai loi vi sinh vt l S. Cerevisiae to ra hm lng ru trong dch ln men v sau , s

dng Leuconostos trong ln men ph qu trnh tng tr, nhm nng cao cht lng ca ru. Ngy nay, ngi ta tin ti dng mt chng vi sinh vt chuyn gen thc hin c hai qu trnh. Cng ngh di truyn ng vai tr quan trng trong vic tuyn chn v to ra cc vi sinh vt c hot tnh enzyme cao, s dng trong cng ngh thc phm. i vi cc sn phm ln men sa nh phomat v sa chua, trc kia, ngi ta thng s dng nhng vi sinh vt t nhin c mt trong sa ln men, do vy, ngi ta kh lng kim sot qu trnh ln men v hiu qu khng cao. Ngy nay, vi cng ngh di truyn, ngi ta to c cc chng mi vi cc tnh cht xc nh v iu khin c qu trnh ln men theo nh hng mong mun. Trong nhng nm gn y, bng cch s dng cng ngh di truyn, ngi ta tuyn chn v to ra nhng chng vi sinh vt c kh nng tng hp cc enzyme chu nhit, chu axit, chu kim tt sn xut enzyme. Enzyme -amylase chu nhit v ang c s dng nhiu sn xut nha, ng glucose t tinh bt. thi gian u, nhng nghin cu ca cng ngh di truyn ch yu hng vo nhng vn sinh hc c bn thun tu v cho n nhng nm gn y, nhnh cng ngh ny chuyn thnh mt nghnh cng nghip tr gi nhiu t USD. Bn cnh nhng ng dng cc k to ln, c li cho con ngi, cng cn nhn nhn s quan tm, lo lng chnh ng v cc thc phm chuyn gen. Tuy ngy nay, cc nh khoa hc c th tng hp c cc gen nhn to hay ghp gen ny hay gen kia vo b my di truyn ca mt sinh vt no , nhng chc chn, cn rt nhiu vn v nhng b n ca s sng, nht l cc sinh vt bc cao vn cha c khm ph. Ngi ta c th chng t s v hi ca cc loi thc phm t thc vt, ng vt hay vi sinh vt chuyn gen qua cc th nghim, nhng v lu di, ngi ta cha th khng nh c s v hi , v vy, khng t ngi t ra lo lng khi s dng chng thng xuyn vi mt s lng ln.

CHNG I
BN CHT CA VT CHT DI TRUYN
1.1- AXIT NUCLEIC - VT CHT DI TRUYN CA MI SINH VT 1.1.1- Thnh phn cu to ha hc ca axit nucleic Axit nucleic c F. Miescher pht hin nm 1869. C hai loi axit nucleic l axit deoxyribonucleic (DNA) v axit ribonucleic (RNA). Axit deoxyribonucleic (DNA) l polyme c phn t lng ln, c mt trong tt c t bo sng. DNA tp trung ch yu cc nhim sc th trong nhn t bo, ngoi ra cn c mt s lng nh nm ty th v lc lp - l cc DNA ngoi nhn. S lng DNA l khng thay i trong nhn ca cc t bo cng loi. Axit ribonucleic c mt c trong nhn t bo v trong nguyn sinh cht 1.1.1.1- Thnh phn nguyn t Trong cu to ca axit nucleic c 5 nguyn t ha hc l carbon (C), hydro (H), oxy (O), nit (N) v phospho (P). Trong , thnh phn nit thng chim khong t 8-10% v phospho l 15-16%. 1.1.1.2- Thnh phn cu to ha hc Phn t axit nucleic c cu to t 3 thnh phn chnh l cc baz nit, ng pentose v axit phosphoric. Khi thu phn hon ton axit nucleic bng enzyme hoc bng axit th thu c ba thnh phn chnh l baz nit, ng pentose v axit phosphoric. Nu thu phn tng bc bng cc enzyme th u tin, enzym ribonuclease ct lin kt phosphoester, gii phng cc nucleotide - l n v cu to c bn ca phn t axit nucleic. Cc nucleotide s tip tc b thu phn di tc dng ca cc enzyme nucleotidase v nucleosidase gii phng axit phosphoric, ng pentose v baz nit (Hnh 1-1).
Tai lieu chia se tai: wWw.SinhHoc.edu.vn Axit nucleic (DNA v RNA)

- Deoxyribonulease - Ribonuclease
Nucleotide
Nucleotidase
Axit phosphoric
Nucleoside
Nucleosidase
ng pentose - Deoxyribose - Ribose
Baz nit - Purine - Pyrimidine
Hnh 1-1: S thu phn tng bc ca axit nucleic 1,- Cc baz nit: C 2 nhm baz nit l purine v pyrimidine (Hnh 1-2): - Baz purine l hp cht nit d vng. Vng purine c nh ho hc c E. Fischer gi ln u tin, trong , bao gm mt vng pyrimidine v mt vng imidazol ghp li. Cc baz c nhn purine l adenine (A) v guanine (G). Mi baz u c 2 dng ng phn. Mt dn xut quan trng ca baz adenine l hypoxanthine. Hypoxanthine c to thnh khi nhm NH2 ca adenine c thay bng nhm OH. Hypoxanthine c ngha quan trng trong qu trnh trao i cht ca t bo sng. - Baz pyrimidine l mt vng 6 cnh c cha hai nguyn t nit. Cc baz c nhn pyrimidine l cytosine (C) v thymine (T). Trong iu kin sinh l, guanine v thymine thng tn ti dng ceton. i khi, baz cytosine cn gp di dng 5-methyl cytosine, cn adenine v cytosine thng tn ti di dng amin.
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2
HN N
5
N N NH
N N
N NH
6 3
7 9
N
8
Adenine (A) (6-aminopurine)
NH HN HN 2
O N N NH HN 2 N
OH N N NH
Guanine (G) (2-amino 6-oxypurine)
NH HN O
3
NH2 N
NH
NH
4 1
5 6
Cytozine (C) (2-oxy-4-aminopyrimidine)
O HN O NH C H3 O N
OH C H3 NH
Thymine (T) (2,4 dioxy-5 methylpyrimidine)
O HN O NH O N
OH
NH
Uracil (U) (2,4 dioxypyrimidine)
Hnh 1-2: Cng thc cu to ca cc baz nit

2,- ng pentose: Trong DNA c cha gc ng deoxyribose, trong RNA cha gc ng ribose nm di dng vng, c cng thc cu to l:
c5h10o5 hoh2c H H H OH H Ho h2c H H H OH H
Pentose
OH H
OH OH
Deoxyribose
1.1.1.3- Nucleoside v nucleotide 1,- Nucleoside:
NH2 N N N N9 Lin kt glycoside
Ribose
O HN O N1
HOH2C H H
O H R
1
HOH2C H R = OH hoc H H H
O H R
1
OH
OH
Hnh 1-3: Lin kt glycoside gia baz nit v ng pentose Khi mt baz nit lin kt vi phn t ng pentose bng lin kt ,N-glycoside s to thnh mt nucleoside. Lin kt ,N-glycoside ny hnh thnh gia nguyn t carbon th nht (C1) ca ng pentose vi nguyn t th 9 (N9) ca baz purine hoc vi nguyn t th nht (N1) ca baz pyrimidine. - Nucleoside ca baz pyrimidine vi ng ribose mang tn ca baz v c ui l -idine (thymidine, uridine, cytidine).

- Nucleoside ca baz purin vi ng ribose mang tn ca baz v c ui l -osine (adenosine, guanosine). - Khi baz nit lin kt vi ng deoxyribose th c thm tip u ng deoxy- (deoxyadenosine, deoxyguanosine, deoxycytisine). Ring baz thymine ch c mt trong DNA nn gc ng trong nucleoside lun l deoxyribose, trong trng hp ny, nhiu khi ngi ta khng cn gi thm tip u ng deoxy-, m ch gi mt cch n gin l thymidine. 2,- Nucleotide:
NH2 N N O HO P OH H H OH O C H2 O H H HO N N O P OH H H OH O C H2 O H H HN O N O
OH
OH
Adenosine 5-monophosphat (Adenosine monophosphat) (AMP)
Uridine 5-monophosphat (Uridine monophosphat) (UMP)
Hnh 1-4: S AMP v UMP Nucleotide l ester phosphat ca nucleoside. Axit phosphoric to lin kt ester vi mt nguyn t carbon no ca ng (thng l C5), to thnh nucleotide (Hnh 1-4). Cc nucleotide l n v cu to ca axit nucleic, phn t DNA c cu to nn t 4 loi nucleotide: dAMP, dGMP, dCMP v dTMP. Nucleotide ng vai tr sinh hc quan trng. Ngoi chc nng cu to nn vt cht di truyn, chng cn c mt trong cc coenzyme, xc tc nhiu phn ng ha hc trong t bo nh cc coenzyme NAD, FAD, FMN v coenzyme A.

Bng 1-1: Tn gi v ch vit tt ca mt s nucleotide Tn gi Adenozine 5-monophosphat (axit adenylic) Guanosine 5-monophosphat (axit guanylic) Cytidine 5-monophosphat (axit cytidytic) Uridine 5-monophosphat (axit thymidylic) Deoxyadenosine 5-monophosphat (axit deoxyadenylic) Deoxyguanosine 5-monophosphat (axit deoxyadenylic) Deoxycytidine 5-monophosphat (axit deoxycytidylic) Deoxythymidine 5-monophosphat (axit deoxythymidylic) 1.1.2- Cu trc phn t DNA 1.1.2.1- c im cu to Axit deoxyribonucleic (DNA) l phn t mang thng tin di truyn ca t bo sng m trong , gen l n v di truyn c bn. DNA c xy dng t 4 loi nucleotide: dAMP, dGMP, dCMP v dTMP. Gc ng trong cc nucleotide ny l deoxyribose. DNA l mt polynucleotide, cc nucleotide ni vi nhau bng lin kt 3',5' phosphodiester. Hai lin kt ester c hnh thnh gia gc phosphat vi carbon th 3 ca nucleotide ny v carbon th 5 ca nucleotide nm k n (Hnh 1-5). Phn t DNA bao gm hai si polynucleotide ngc chiu nhau, baz purine ca si polynucleotide ny nm i din vi baz pyrimidine ca si polynucleotide kia theo quy lut b sung nghim ngt: adenine ng i din vi thymine (AT) v guanine ng i din vi cytosine (G-C). Hai si polynucleotide c gi vng v n nh nh cc lin kt hydro gia cc baz nit ca hai mch (Hnh 1-5). K hiu AMP GMP CMP UMP dAMP dGMP dCMP dTMP

H N H N H N N H N H H N N N N
1N
7 N 8 9 N
R
N 3
N
R
N
R
H N
H N H
H H
H O N N
R
4 3N 2 1
5 6
O
N N
R
Dng amin
Dng imin t gp (2 dng quay)
H O H N H N
H O
N
O
N
6
N
N H N
H N
N H N H N
N R
N R
N
R
H O N O CH3 N
R
H O CH3 N O N
R
CH3
O H N O
4
N
R
O N CH3
H
O N O N
R
CH3
Dng ceton
O H N
R
Dng enol t gp (2 dng quay)
Hnh 1-5: V tr c th hnh thnh lin kt hydro ca cc dng baz nit

Hng chuyn dch in t trong lin kt hydro

Quy lut lin kt b sung gia hai loi baz nit do E. Chargaft pht hin. Khi nghin cu thnh phn cc baz nit trong phn t axit nucleic, ng thy rng: S baz adenine lun bng thymine v s baz guanine lun bng cytosine. Quy tc ny c gi l quy tc Chargaft: A = T v G = C hay A+G T+C =1
V sau, cc nh nghin cu thy rng: s lng cc cp baz AT v GC rt khc nhau mi loi nn quy tc ny c b sung ni dung sau:
T l (A+T)/(G+C) l tu theo loi
Mt c im rt quan trng ca cc baz nit nh nu phn trn l chng c cc dng ng phn. Nu xt v gc ho hc thun tu, da vo kh nng cho v nhn in t ca cc nguyn t trong cc loi baz nit (Hnh 1-5), th khi thay i dng ng phn, baz adenine c th to lin kt hydro vi c baz thymine (AT) v cytosine (AC), tng t nh vy, baz guanine cng c th to lin kt vi cytosine (GC) v thymine (GT). Trong iu kin sinh l ca t bo, ngi ta thy, baz adenine v cytosine thng nm di dng amino, ngha l, nguyn t nit gn vi vng purine v pyrimidine lun c hai nguyn t nit (NH2), rt t khi gp dng imin (NH). Tng t nh vy, baz guanine v thymine, nguyn t oxy gn carbon th 6 ca vng purine v pyrimidine lun nm di dng ceton (C=O) v rt t khi gp dng enol (COH) (Hnh 1-5). Nh vy, v tr ca nguyn t hydro cc nhm th trn l ht sc quan trng. Nu nguyn t hydro khng c nh v tr th s dn n s bt cp nhm gia AC v GT, lm thay i trnh t sp xp v thnh phn cc baz nit trn cc si polynucleotide. Nu trng hp ny xy ra th b my di truyn s b bin ng, phn t DNA ca th h ny s khc th h trc. Tuy nhin, t bo c th sng lun c cch kim sot thch hp m bo b my di truyn n nh t th h ny qua th h khc, v vn ny, chng ta s xem xt phn sau.

1.1.2.2- Cu trc bc II - M hnh Watson v Crick Nm 1953, Watson v Crick khm ph ra m hnh cu trc phn t DNA - y l mt pht minh quan trng ca th k XX, nh du mt bc ngot cho s pht trin ca di truyn hc. Watson v Crick c trao gii thng Nobel nm 1962. Theo Watson v Crick, m hnh cu to khng gian ca DNA c nhng c im chnh sau: - Phn DNA gm hai si polynucleotide sp xp theo hai hng ngc chiu nhau (i song song): si bn ny c u 3'OH th si bn kia s l 5'P. - Hai si cng xon (xon i) xung quanh mt trc chung. - Cc baz nit ca hai si nm quay vo trong. Baz ca si ny ng i din vi baz ca si kia theo quy lut b sung A i din T v G i din C. A ni vi T bng hai lin kt hydro, G ni vi C bng 3 lin kt hydro. - Nhm phosphat v gc ng trong chui polynucleotide xoay ra ngoi, hnh thnh lin kt vi nc, m bo tnh n nh cho phn t. - Mi vng xon c tng ng vi 10 cp baz, chiu cao ca mi vng xon c l 34 (1 = 10 m). Nh vy, chiu cao ca mi nucleotide l 3,4, ng knh trong l 20 (Hnh 1-6). 1.1.2.3- Cc dng cu trc ca DNA Phn t DNA sinh vt eucaryote c dng thng, cn phn ln t bo procaryote c dng vng. Tuy nhin, d vng hay thng, cc DNA u c cu to cun xon. + Dng thng: Phn t gm hai si xon kp, i song song, mi si u c u 3'OH v 5'Phosphat t do.
-10
3 5
H O O P O CH2
4
O
2 3
H O N N O H H H
O N N N H
N H N
1 H
H O
H
4
Guanine
Ch2
H O O P O
H 1
N O H
3 2
Cytosine
H O N CH2 H O H
3
O O P O
3
H N N N H H H N H H
ch3
H H
N N O
H O
Thymine
CH2
Adenine
O O P O O
3 5
Hnh 1-6: Cu to ca 2 si polynucleotide v s bt cp b sung ca cc baz nit

P 5' 5' 5' P 5' 3' P 5' P 5' OH 3' 3' 3' 3' P 3' 3' OH 3' 5' P 5' P 5' P 5' P 5' P 5' 5' 3'
A C T G A
T G A C T
3' 3' 3' 3' 3'
Hnh 1-7: M hnh cu trc phn t DNA
0,34 nm (3,4 A)
3,4 nm
(34 A)

Ngy nay, ngi ta pht hin v m t c 6 loi cu trc xon i ca DNA l A, B, C, D, E v Z. S khc nhau gia cc loi c th hin ch yu nhng c im sau: - Chiu xon (xon phi hoc xon tri), - S lng i baz trong mi vng xon, - Khong cch gia mi i baz, - Khong cch ln nht gia mi si. Loi B l loi hay gp nht trong iu kin sinh l v l loi ng theo m hnh ca Watson v Crick, c chiu xon phi. Loi Z c tm thy trong nhim sc th ca rui dm, c chiu xon tri v 12 i baz trong mi vng xon. Loi A c tm thy trong mi trng cha nhiu ion natri hay canxi, c chiu xon phi v c 11 i baz trong mi vng xon. Loi C, D v E khng c mt trong c th sng. + Dng vng: Phn t hnh trn, xon. C th gp dng xon n vng nh DNA v mt s virus hay dng xon i ca DNA vi khun. 1.1.3- Tnh cht ca DNA Dung dch DNA c tnh keo do phn t ln v c tnh axit do c cha gc axit phosphoric. Di tc dng ca cc tc nhn nh nhit hay cc cht ha hc (formamide, ur), hai si n ca phn t DNA b tch ri do cc lin kt hydro gia cc baz b sung b ph v - hin tng ny gi l s bin tnh ca DNA. Gi tr trung bnh ca khong nhit trong qu trnh bin tnh gi l nhit nng chy ca DNA (Tm - melting Temperature). Sau khi hai mch n ca phn t DNA tch ri ra, nu ta gim nhit t t, cng vi iu kin thch hp th hai mch s bt cp tr li - hin tng ny gi l s hi tnh. Nu ta gim nhit mt cch t ngt th s bt cp tr li s khng din ra.

Cc nucleotide trong DNA hp th tia cc tm vi di bc sng ti a l 260nm. Do vy, kh nng hp th tia cc tm ca hai si n s ln hn mt si kp. DNA b thy phn di tc dng ca cc enzyme nuclease. 1.1.4- Cc th nghim chng minh DNA l vt cht di truyn 1.1.4.1- Cc chng minh gin tip Trc y, ngi ta cho rng, protein l vt cht di truyn, quan nim ny vn cn tn ti cho n nhng nm u ca th k XX. Sau khi pht hin ra axit nucleic (1869) v nh ha hc c R. Feulgen tm ra phng php nhum mu c hiu vi axit nucleic (1914), th rt nhiu kt qu nghin cu v axit nucleic lm sng t rng, DNA mi l vt cht di truyn ch khng phi l protein. Nhng pht hin sau y gin tip cho thy DNA l vt cht di truyn: - DNA c mt trong tt c cc t bo sng, t vi sinh vt, thc vt cho n cc ng vt bc cao. - DNA l thnh phn ch yu ca cc nhim sc th trong nhn t bo. - Hm lng DNA trong tt c cc t bo dinh dng (t bo soma) ca mt loi sinh vt bt k no u ging nhau, khng ph thuc vo trng thi hay chc nng ca chng. Ngc li, hm lng RNA v protein li thay i ty theo trng thi sinh l. - Khi gy t bin bng tia t ngoi, ngi ta thy hiu qu gy t bin cao nht l bc sng 260nm, l bc sng m DNA hp th cao nht. - S lng DNA trong cc t bo sinh dc (trng, tinh trng, non, phn hoa, ...) bng mt na s lng DNA trong t bo dinh dng ca c th. 1.1.4.2- Th nghim ca Griffith v Oswald Avery Nm 1928, Griffith pht hin ra hin tng bin np (transformation) vi khun Streptococcus pneumoniae gy bnh sng phi ng vt c v. Vi khun ny c hai dng khc nhau:

- Dng S (Smooth) c khun lc lng trn mi trng thch. T bo ca vi khun dng ny c v bao (capsule) nn h thng min dch ca c th ng vt khng th tn cng tiu dit c, v vy, khi xm nhp vo c th, chng gy nn bnh sng phi. - Dng R (Rough) c khun lc nhn, t bo ca chng khng c v bao, nn khi xm nhp vo c th ng vt, chng s b h thng min dch ca ng vt tiu dit, khng gy nn bnh. Griffith pht hin ra rng, nu tim dch vi khun dng S un si n cht vo chut th chut khng b bnh. Nhng khi tim vo chut hn hp bao gm mt lng nh vi khun sng dng R vi mt lng ln t bo vi khun dng S un cht, th chut pht bnh v cht. Ly mu ca chut cht v bnh ny a vo mi trng nui cy, ng thy s c mt ca vi khun dng S. Nh vy, vi khun dng S khng th t sng tr li sau khi b un n cht c, nhng cc t bo cht ny truyn tnh gy bnh cho t bo sng dng R. Hin tng ny gi l bin np. Nm 1914, Oswald Avery, Colin Mc. Leod v Maclyn Mc. Carty xc nh tc nhn gy bin np bng th nghim theo s nh Hnh 1-8.
me zy e n tein g n pro b hn l X y p h t
Dng R v dng S T bo vi khun dng R
Tch DNA
T bo vi khun dng S
DNA + mt s protein
X th l b y p ng h en n D zy NA m
Ch c dng R T bo vi khun dng R
Hnh 1-8: S th nghim ca Oswald Avery, Colin Mc. Leod v Maclyn Mc. Carty

DNA ca t bo vi khun gy bnh dng S c tch v lm sch. Mc d tch v lm sch nhng sn phm thu nhn c vn cn mt t protein. Gi thit, nu protein l tc nhn gy bin np th sau khi x l loi b protein bng enzyme protease v phi trn vi t bo sng dng R, th hin tng bin np s khng xy ra. Ngc li, nu tc nhn bin np l DNA th sau khi loi b DNA bng enzyme deoxyribonuclease v phi trn vi t bo sng dng R th cng s khng xut hin hin tng bin np. Kt qu th nghim cho thy, hin tng bin np ch tm thy khi c mt DNA, cn trng hp DNA b enzyme ph hy th khng xut hin hin tng bin np. iu khng nh rng, chnh DNA l tc nhn gy bin np, truyn tnh gy bnh t t bo dng S sang t bo dng R ca vi khun. 1.1.4.3- Th ngim ca A. Hershey v M. Chase Nm 1952, bng th nghim v s xm nhp ca virus bacteriophage T2 (gi tt l phage) vo vi khun E. Coli, Alfred Hershey v Martha Chase chng minh trc tip rng, DNA chnh l vt cht di truyn.
Protein DNA Tr ui ui a gc Lng ui
32
35
bm vo
Hnh 1-9: S v s xm nhp ca virus phage T2 Phage T2 c cu to gm 2 thnh phn chnh, v protein bn ngoi v DNA bn trong phn u, t l gia 2 thnh phn ny l tng ng. Khi xm nhp vo vi khun, ngi ta xc nh rng: u tin, phn ui ca phage bm vo mng t bo ca vi khun, sau , mt phn cht no c bm

vo t bo vi khun v sau mt thi gian, rt nhiu t bo virus mi c to thnh bn trong t bo ca vi khun v chui ra ngoi. Th nghim ca A. Hershey v M. Chase c tin hnh vi mc ch xc nh xem, cht no ca phage c bm vo t bo vi khun to ra cc th h phage mi. Da vo thnh phn cu to ca DNA v protein, ngi ta thy rng: DNA cha nhiu phospho nhng khng cha lu hunh, cn protein th cha lu hunh. V vy h s dng 2 ng v phng x l S
35
v P
32
gn vo
protein v DNA ca phage T2 nhm d dng theo di. Tin trnh th nghim gm cc bc sau: - To ra mt th h phage T2 c protein cha S T2 mi to thnh s mang ng v phng x S - Tch virus mang ng v phng x. - Cho virus mang ng v phng x xm nhp vo t bo vi khun E. Coli khng mang ng v phng x. Bc ny thc hin bng cch cho phage T2 tip xc vi t bo vi khun trong khong thi gian virus bm vo mng t bo ca vi khun v bm vt cht di truyn ca chng vo trong t bo, ri dung dch c lc mnh v li tm tch ri t bo vi khun ra khi phn cn li ca phage bn ngoi t bo. - Phn tch thnh phn phng x phn nm ngoi t bo vi khun ca phage v phn nm bn trong t bo vi khun. Kt qu cho thy: Phn nm ngoi t bo vi khun ca phage c cha nhiu S (80%) nhng rt t P . iu cho thy rng, phn ln protein ca v phage nm ngoi t bo vi khun. Ngc li, phn bn trong t bo vi khun c cha nhiu P (70%), nhng t S , chng t rng, DNA c bm vo t bo vi khun sinh sn ra mt th h phage mi. T cc kt qu th nghim i n kt lun: Vt cht di truyn ca phage T2 l DNA.
32 35 35 32 35 35 35
v c DNA cha P
32
32
bng cch nui vi khun E. Coli trn mi trng c S

32
v P . Th h phage
v P .

1.2- CC RNA 1.2.1- Cu to ha hc v c im chung ca RNA Axit ribonucleic (RNA) cng l mt axit nucleic. Hai c im c bn v cu to, phn bit gia axit ribonucleic v axit deoxyribonucleic l cu t ng v baz nit. Trong phn t RNA c cha ng ribose, cn trong DNA c cha ng deoxyribose. Thnh phn cc baz pyrimidine trong RNA v DNA cng khc nhau. DNA cha cytosine v thymine, khng bao gi c uracil, ngc li, RNA cha cytosine v uracil, khng khi no c thymine. Ngoi ra, trong RNA c nhiu baz th yu (cc baz nit t gp) hn trong DNA. n v c bn xy dng nn RNA cng l cc nucleotide. Cc nucleotide lin kt vi nhau cng bng lin kt 3'-5'-phosphodiester to thnh phn t polynucleotide nh DNA. V cu trc phn t, RNA c cu trc mch n ch khng phi l mch kp nh DNA. Cu trc bc I ca RNA xc nh trt t sp xp cc gc nucleotide trong chui polynucleotide.
Hnh 1-10: M hnh biu din kh nng to thnh cu trc xon i nhng on RNA c cc trnh t baz b sung Cu trc bc II ca RNA cng l xon nhng khc vi DNA, phn t RNA l mt chui polynucleotide lin tc nn cu to xon ch thc hin trong phm vi mt phn t. Khi , chui polynucleotide to cu trc xon bng cch to nn cc lin kt hydro gia adenine v uracil (AU) v gia guanine v cytosine (GC). Trong cu trc bc II ca RNA, ch c khong 50% chui polynucleotide ca phn t RNA c xon, cn cc phn khc th khng. Hn na, cu hnh ca cc on xon cng cha hon thin nh DNA. Do khng c s tng ng hon ton trong trt t ca cc baz b sung nn mt s mt xch nucleotide ring l c dng vm li.

RNA nm trong bo tng, trong ribosome v c trong nhn t bo, nhng tp trung ch yu trong bo tng. 1.2.2- Cc loi RNA 1.2.2.1- RNA thng tin (mRNA) RNA thng tin (messenger RNA) c tng hp trong nhn t bo trn khun ca DNA nn chng cha mt lng ln thng tin cn thit cho s tng hp cc protein c hiu khc nhau. ln ca mRNA ph thuc vo ln ca protein cn tng hp, nh vy, cc phn t mRNA ca mt t bo c kch thc rt khc nhau v trnh t nucleotide ca cc RNA thng tin cng rt khc nhau. Sau khi c tng hp nhn t bo, mRNA s chuyn t nhn n ribosome, mang nhng thng tin cn thit cho qu trnh tng hp protein. S cu trc chung ca cc phn t mRNA c th chia lm ba on: u 5 c on dn u, tip sau l on m ha protein v on theo sau, on dn u v on theo sau khng mang m cho cc axit amin.
Hnh 1-11: S cu trc chung ca phn t mRNA 1.2.2.2- RNA vn chuyn (tRNA) tRNA (transfer RNA) lm nhim v vn chuyn cc axit amin c hot ha n ribosome l ni tng hp nn phn t protein. tRNA chim khong t 10% n 20% tng lng RNA ca t bo, v c trng lng phn t khng ln lm, n ch cha khong t 75 n 90 nucleotide. Ngy nay, ngi ta xc nh c thnh phn v trt t sp xp ca hn 100 tRNA. Mi axit amin trong 20 axit amin, c th kt hp vi mt hoc mt s dng tRNA. im khc trong cu to ca tRNA l, ngoi bn baz nit thng thng l A, G, C v U, n cn cha mt lng nh cc baz b sung ph nh 6-methylaminadenine, dimethylguanine, inosine, ...

A C C A C C U G C U G U C A G G C C
Alanine I D T 1 mG m22G 1 mI Inosine Dihydrouridine Ribothymidine Methylguanosine Dimethylguanosine Methylinosine
5' G G G C G U m1G G D C G A U G C G C G
G D A
G C G C m2 G 2 C U C C C U U
U C C G G C D G A G A Cc baz ngoi lai G Pseudouridine Y G CCG Codon G
I C C
I G C m1 I 5' mRNA
Anticodon
3'
C C G
Hnh 1-12: M hnh cu to RNA vn chuyn alanine ca nm men C th tm tt mt s im chnh v cu to ca phn t tRNA nh: - u 3 ca phn t lun kt thc bng b ba CCA, cn u 5-(nhm monophosphat) thng kt thc bng gc axit guanilic (G). mi phn t thng c bn on c cha cc lin kt hydro gia cc baz b sung v 4 vng li, , gia cc nucleotide khng c lin kt hydro v cc baz khng c cp b sung. - u 3 kt thc bng 3 nucleotide CCAOH. Phn t aminoaxit lun lun gn u 3 baz A cui.

O H H C N Ribose N
C C O
N
C C
H N Ribose
N N
C N C
N C N
N N N H
Ribose
Ribose
Inosine-Cytosine Anticodon Codon Codon hoc anticodon

O C H C N Ribose N C C N C H C N H O O H N C N Ribose C C H H C N N C C N C H C N O H
Guanine-Uracil Codon hoc anticodon

H H N C H N C H N C C N C N H
Ribose
Ribose
Inosine-Uracil Anticodon Codon
Inosine-Adenine Anticodon Codon
U CU 5 AGA 3
Anticodon Si mRNA 5
U CU AGA 3
Codon
Codon
Baz U ca anticodon c th lin kt vi baz A hoc G ca codon
CC I 5 G GU 3
Anticodon Si mRNA 5
CC I G GC 3 5
CC I GGA 3
Codon
Codon
Codon
Baz I ca anticodon c th lin kt vi baz U, C hoc A ca codon
Hnh 1-13: S hnh thnh lin kt hydro gia nucleotide th 3 trong codon vi baz ngoi lai v baz khng nm theo quy lut (AU, GC) trong anticodon ca phn t tRNA

- Vng li nm gn k u 3 (Hnh 1-12) cha 7 baz khng sp xp theo quy lut b sung, nn gia cc baz khng c cc lin kt hydro. Hin nay, ngi ta cho rng, RNA vn chuyn gn vi b mt ca ribosome nh vng li ny. - Vng li k tip (tnh t u 3) vi ln rt tht thng, gi l vng li ph (extra loop). - Vng li th ba (Hnh 1-12) cha 7 baz khng c lin kt hydro (khng b sung), trong c 3 baz ch yu k nhau - anticodon l b ba i m, b ba ny s b sung cho b ba m ha (codon) trn RNA thng tin. Nh vy, cc axit amin c a n ng v tr trong qu trnh tng hp protein. tRNA vn chuyn alanine nm men v tr th ba ca b i m (anticodon) l baz inosine (I) l dn xut ca purine, inosine c th to lin kt b sung vi c ba loi baz l A, U v C (Hnh 1-13). - Vng li tip theo (th t) cha 8 n 12 baz khng b sung, gi l vng li D (D-loop). 1.2.2.3- RNA ribosome (rRNA) Ribosome c cu to t hai tiu n v, gm mt tiu n v ln v mt tiu n v nh. Mi tiu n v c xy dng t ba thnh phn chnh l protein, lipit v rRNA. C ba loi phn t rRNA thng gp trong t bo vi khun, l: - rRNA c di 1542 nucleotide (16S) trong tiu n v nh ca ribosome, - rRNA c di 2904 nucleotide trong tiu n v ln (23S), - rRNA rt nh, c di 120 nucleotide (5S) cng nm trong tiu n v ln. - Trong t bo sinh vt eucaryote, tiu n v nh ca ribosome cha si rRNA 18S, cn tiu n v ln cha hai si rRNA l 28S v 5,8S. Trong c ba loi rRNA u c s lng cc baz b sung khng bng nhau (GC v AU).

1.2.2.4- Nhim sc th Nhim sc th c th quan st r di knh hin vi k gia ca phn bo nguyn nhim. Nhim sc th c cu trc phc tp, c bit l nhim sc th ca cc vi sinh vt nhn chun (eucaryote). Trong mt nhim sc th c mt phn t DNA si kp cun xon, lin kt vi protein histon. Mi nhim sc th c hnh dng c trng ring, hnh dng c th quan st r trong thi k phn bo. T bo sinh vt n bi c mt b nhim sc th (n NST), v d nh vi khun c mt nhim sc th. Sinh vt lng bi nh con ngi v ng vt, t bo c hai b nhim sc th (2n NST). Sinh vt lng bi thng sinh sn hu tnh nn trong c th, ngoi t bo dinh dng (t bo soma) cn c t bo gii tnh. t bo dinh dng lun c 2n nhim sc th, cn t bo gii tnh ch c n nhim sc th. cc t bo lng bi (2n NST), mi nhim sc th c mt cp ging nhau, gi l nhim sc th tng ng. S lng nhim sc th trong tt c cc t bo dinh dng ca mt c th sng u ging nhau, cho d chc nng hot ng ca cc c quan trong c th sng khc nhau. S lng nhim sc th l c trng cho tng loi, v d: - Ngi : 2n = 46 nhim sc th,
- Rui dm: 2n = 8 nhim sc th, - Vt: - Nga: 2n = 80 nhim sc th, 2n = 64 nhim sc th.
S lng nhim sc th khng mang ngha cho s tin ha. Nhim sc th nhn r nht di knh hin vi vo k gia ca nguyn phn, khi cc mch xon v co ngn mt cch cc i. Mi nhim sc th c mt im tht eo gi l tm ng (centromere). Tm ng chia nhim sc th lm hai phn vi chiu di khc nhau. Da vo v tr tm ng c th phn bit ba kiu hnh thi ca nhim sc th: - Nhim sc th cn tm: Tm ng nm gia nhim sc th.

- Nhim sc th lch tm: Tm ng chia nhim sc th lm hai phn khng u nhau, mt bn di v mt bn ngn. - Nhim sc th mt tm: Tm ng nm gn cui ca mt u, chia nhim sc th lm hai phn rt khng u nhau, mt bn rt di v mt bn rt ngn. Bng phng php nhum mu v quan st di knh hin vi quang hc giai on khng phn bo, ngi ta nhn thy trn nhim sc th c vng nhum mu m v c vng nhum mu rt t. Vng bt mu m gi l cht d nhim sc (heterochromatine) v vng t bt mu thuc nhum gi l cht nguyn nhim sc (euchromatine). Ngy nay, bng nhng thit b nghin cu hin i, ngi ta chng minh rng, trng thi cun xn cao ca phn t DNA v protein, nhim sc th s bt mu tt vi thuc nhum, cn trng thi gin xon th s bt mu km hn. Nh vy, cht d nhim sc l trng thi cun xon, khng phin m c ca DNA, cn cht nguyn nhim sc l trng thi DNA ang hot ng v gin xon. 1.3- M DI TRUYN 1.3.1- Khi nim v m di truyn Chng ta bit rng, trnh t cc baz nit trn DNA quyt nh trnh t ca axit amin trn protein tng ng. Tt c c 20 axit amin trong protein, nhng ch c 4 loi baz nit trong DNA. Nh vy, nu mi baz nit xc nh mt axit amin, th ch c 4 axit amin c xc nh. Nu c hai baz nit xc nh mt axit amin, th s lng cc axit amin c xc nh ch l 4 =16, cn thiu 4 axit amin cha c xc nh. Vy, ti thiu phi 3 baz nit xc nh mt axit amin. Nh th, s t hp b ba c th c t 4 baz l 4 =64. Nu m di truyn l nhng b ba th xy ra trng hp nhiu b ba xc nh mt axit amin. Ngy nay, bng kt qu thc nghim, ngi ta chng minh rng, b ba m di truyn l ng. Trong b m di truyn, mt b ba nucleotide c gi l codon.
3 2

1.3.2- c tnh ca m di truyn M di truyn c mt s c tnh sau: 1,- M di truyn khng c du phy, ngha l thng tin di truyn c c theo tng cm 3 nucleotide mt cch lin tc, khng ngt qung. 2,- Thng tin c c theo mt chiu, bt u t mt im xc nh. Bng 1-2: Bng m di truyn
V tr th hai V tr th nht - u 5'
U U
UUU UUC UUA UUG CUU CUC CUA CUG UCU UCC UCA Leu UCG Phe CCU CCC Leu CCA CCG ACU ACC ACA ACG
C
Ser
A
UAU Tyr UAC UAA* Stop UAG* Stop CAU CAC CAA CAG AAU AAC AAA AAG GAU GAC GAA GAG His Gln Asn Lys Asp Glu
G
UGU Cys UGC UGA* Stop UGG Trp CGU CGC CGA CGG AGU AGC AGA AGG GGU GGC GGA GGG U C A G U C A G U C A G V tr th ba - u 3'
Pro
Arg
AUU AUC Ile AUA AUG* Met
Ser Arg
Thr
GUU GCU GUC GCC Val GUA GCA GUG* GCG
Ala
U Gly C A G
3,- M di truyn mang tnh ph bin, ngha l tt c mi sinh vt u dng chung mt loi thng tin. V d: Mt gen ly t t bo ng vt m ha cho mt loi protein no . Gen d c dch m trong t bo ng vt hay dch m trong t bo E. Coli, th cng ch tng hp nn mt loi protein m thi. 4,- M di truyn mang tnh thoi ha (degenerate), ngha l, nhiu b ba cng xc nh mt axit amin, tr hai ngoi l l b ba AUG m ha cho methionine v UGG m ha cho trytophan.

V d: Valine c 4 b ba m ha cho n l GUU, GUC, GUA v GUG. S khc nhau ch yu ca cc b ba ny l nucleotide th ba. Tuy vy, khng phi lc no cp nucleotide u ging nhau cng m ha cho mt axit amin, v d trng hp ca leucine c 6 b ba m ha, trong c hai b ba c hai nucleotide u khc vi 4 b ba kia, l UUA, UUG v CUU, CUC, CUA, CUG. 5,- M di truyn c nhng b ba khi u v kt thc c hiu. AUG l tn hiu khi u, nu n khng c u 5 ca RNA thng tin th qu trnh dch m khng bt u c. Cc b ba kt thc l UAG, UAA v UGA. 1.4- VT CHT DI TRUYN CA MT S NHM SINH VT 1.4.1- Vt cht di truyn ca virus Virus c pht hin vo cui th k 19, c kch thc rt nh, sng k sinh trn cc loi sinh vt khc nhau, khng c cu to t bo hon thin, ngha l khng c mng t bo, khng c cc bo quan. Virus khng c th t tng hp protein khi chng nm bn ngoi t bo ch, nn sau khi hnh thnh, chng khng tng v kch thc. Cu to ca virus n gin gm mt b my di truyn ca n, gi trong v protein, bn ngoi c th c mng bao. B my di truyn ca virus ch c mt loi axit nucleic: DNA (mch kp hoc mch n) hoc RNA. Cu trc ca phn t axit nucleic trong virus c th dng thng hoc dng vng. B gen nh nht c 4 gen, ln nht c chng vi trm gen. Virus ch c th t ti bn b my di truyn ca mnh v tng hp protein bao gi b my di truyn trong t bo ch. 1.4.2- Vt cht di truyn ca vi khun Vi khun l loi vi sinh vt c cu trc t bo nhng n gin, tc l chng c mng t bo, nguyn sinh cht v cc bo quan, tuy nhin, chng cha c mng nhn. B my di truyn ca vi khun nm trong nguyn sinh cht l phn t DNA mch kp vng trn, c gi l nhim sc th. Ngoi nhim sc th, trong t bo vi khun cn c plasmid - l nhng phn t DNA nh, mch vng, xon kp, chng c kh nng phn chia c lp khng ph thuc vo DNA nhim sc th.

Vi khun thuc nhm t bo n bi - c s nhim sc th l n, cc t bo lng bi lun c s lng nhim sc th l 2n. B my di truyn ca vi khun E. Coli c nghin cu k nht. E. Coli c mt nhim sc th (chromosome), b gen c khong 2.000 n 4.000 gen trn phn t DNA vng trn, xon kp bao gm khong 4,5 triu nucleotide, cu trc khng gian nm di dng siu xon. 1.4.3- Vt cht di truyn ca eucaryote 1.4.3.1- DNA trong nhn T bo ca cc sinh vt c nhn in hnh (eucaryote) cu to hon thin, bao gm mng t bo, nguyn sinh cht, cc bo quan v nhn t bo. Khc vi t bo vi khun, nhn t bo c mng nhn bao bc. B my di truyn ca t bo l nhng phn t DNA nm trong cu trc nhim sc th. Mi nhim sc th cha mt phn t DNA thng, mch xon kp. Cc nhim sc th phn b trong nhn. S lng v hnh dng ca nhim sc th l c trng cho mi loi sinh vt. Cc t bo dinh dng ca nhm sinh vt eucaryote u c s lng nhim sc th l 2n (t bo lng bi). T bo gii tnh ch c mt b nhim sc th n (t bo n bi).
Histone H2A, H2B, H3 v H4
Hnh 1-14: S cu to ca nucleosome Cc nhim sc th ca eucaryote c t chc phc tp. Mi nhim sc th cha mt phn t DNA di mch xon kp lin kt vi protein histone v cun li, to thnh phc hp nucleoprotein.
55A on lin kt
110A

Histone l nhng protein c phn t nh cha nhiu axit amin mang in tch dng nh lysine, arginine nn d lin kt vi phn t DNA mang in tch m. Phn t DNA trong nhim sc th c cun xon nhiu mc khc nhau. Nucleosome l n v cu trc c bn, c to nn do mt on DNA khong 200 cp nucleotide lin kt vi 5 loi protein histone l H1, H2A, H2B, H3 v H4. Cu to ca mi nucleosome gm mt on DNA khong 145 cp nucleotide qun quanh mt ci li gm 8 protein histone l 2H2A, 2H2B, 2H3 v 2H4 v mt on DNA lin kt c di khong 55 cp nucleotide, on ny gn vi protein histone H1. di ca on DNA lin kt ny thay i ty theo chng loi sinh vt. Cc nucleosome xp st vo nhau, to thnh si chromatin. Si chromatin cun xon, un khc nhiu ln, to thnh vng xp cun dy m c, gi l cht d nhim sc. Cht d nhim sc l trng thi cun xon cao ca phc hp nucleoprotein thi k phn bo (Hnh 1-15). Cc nhim sc th c nhn i ln trc thi k phn bo v sau , chia u v cho cc t bo con trong thi k phn bo. Nh vy, cc thng tin di truyn c truyn t th h ny sang th h khc.

A
B
Nucleosome
C
Si Chromatin
Vng xp cun
E
Cht d nhim sc
F
Nhim sc th
Hnh 1-15: Cch sp xp ca phc hp nucleprotein, to thnh nhim sc th giai on phn chia t bo

1.4.3.2- DNA ngoi nhn C mt s bo quan trong t bo cht nh ti th v lc lp ca t bo eucaryote c cha DNA. Cc DNA ngoi nhn ny cng mang gen m ha cho protein v truyn t th h ny sang th h khc, tuy nhin, s phn ly ca cc gen t bo cht khng tun theo cc nh lut ca Mendel v c ch phn chia t bo cht v cc t bo con khng u nh cc nhim sc th trong nhn. Lc lp v ti th l hai loi bo quan tham gia trc tip vo qu trnh chuyn ha nng lng ca t bo. phn bit vi DNA nhim sc th, DNA ca lc lp c k hiu l cpDNA (chloroplast DNA) v DNA ca ti th c k hiu l mtDNA (mitochondrial DNA). B gen ca cpDNA m ha cho cc protein trong thnh phn ca lc lp. cpDNA c cu trc dng vng trn, si xon i c kch thc nh, khong t 120 n 200kb (kilobaz) ty theo chng loi thc vt. DNA ca ti th (mtDNA) m ha cho nhiu protein ca mng bn trong ti th v mt s protein tham gia vo chui chuyn vn in t. mtDNA cng c cu trc dng vng trn nhng nh hn cpDNA nhiu ln. Mc d b my di truyn ngoi nhn l cc DNA ca ti th v lc lp c th t nhn i mt cch c lp v c b my tng hp protein ring, nhng hot ng ca chng c s phi hp cht ch vi b my di truyn trong nhn, hot ng ca cc gen ngoi nhn gp phn b sung cho cc hot ng ca cc gen trong nhn. Nh vy, trong cc loi t bo eucaryote c cha lc lp th tn ti ba loi DNA l DNA nhim sc th, cpDNA v mtDNA, cn cc t bo khng c lc lp th ch c hai loi l mtDNA v DNA nhim sc th.

CHNG II
HOT NG V S BIU HIN CA GEN
2.1- GEN L N V CHC NNG CA B MY DI TRUYN 2.1.1- Cc quan nim v gen Thut ng "gen" c Johansen nu ra vo nm 1909 ch nhn t di truyn xc nh mt tnh trng no . V tr v cu trc ca gen c Morgan v cc nh khoa hc xc nh v ngy cng lm r thm. Giai on trc nm 1953, khi cu trc ca phn t DNA cha c khm ph th cu to ca gen cng cha c xc nh, tuy nhin cc nh khoa hc khng nh rng, gen l mt n v di truyn, mi mt gen xc nh mt tnh trng, c mt v tr nht nh trn nhim sc th v gen c th phn chia nh v mt ti t hp v t bin. Nm 1941, Beadle v Tatum xc nh rng, gen kim tra hot tnh ca enzyme v nu ra gi thuyt l: mi gen kim sot s tng hp mt enzyme. Gi thuyt ny v sau c m rng v c th ha hn l: mi gen kim sot s tng hp mt chui polypeptide. V d, mt phn t enzyme c cu to t hai si polypeptide th s do hai gen xc nh. Sau khi Watson v Crick xc nh c cu trc DNA th cu to ca gen cng ngy cng c lm r hn. Hin nay, cu to v chc nng ca mt gen c xc nh nh sau: - Gen l mt on DNA m bo cho vic to ra mt polypeptide hay mt RNA. Mi gen c cu to gm ba vng (region) c chc nng ring bit l: vng trc, vng sau v vng m ha. Vng trc lm nhim v iu hnh, vng trc v vng sau khng m ha cho cc axit amin. Trong vng m ha t bo sinh vt eucaryote c nhng on mang m cho cc axit amin gi l exon xen k vi nhng on khng mang m gi l intron. Mi gen chim mt v tr (locus) nht nh trn nhim sc th v xc nh mt tnh trng nht nh. Gen c th b chia nh bi cc n v t bin v ti t hp.

2.1.2- Cu trc ca gen Mi mt gen c cu to tng qut gm ba vng chnh: vng iu khin (vng trc), vng mang thng tin di truyn (vng m ha) v vng kt thc (vng sau). Vng iu khin nm u 3 v vng kt thc nm u 5 ca si DNA lm khun (coding strand hoc sense) phin m. Tuy nhin, khi ghi s ca mt gen bt k no vo ngn hng gen, ngi ta u ly si DNA khng lm khun (antisense) v trnh t sp xp cc baz trong si antisense ging nh trnh t sp xp cc baz trong si RNA sau khi phin m. Do , c th ni vng iu khin nm u 5 v vng kt thc nm u 3 ca gen. Vng iu khin v vng kt thc khng mang m cho cc axit amin trong qu trnh tng hp protein. Ngoi ra, trong mt gen cn c th c mt s cu trc c th khc, c v tr khng xc nh nh trnh t tng cng (enhance), trnh t bt hot (silencer),... S cu trc chung ca mt gen c th biu din nh sau:
vng mang thng tin di truyn
5'
P
vng iu khin
3' vng kt thc
R: Trnh t iu ho P: Promoter O: Operator Vng iu khin khng c phin m m c chc nng gip enzyme RNA-polymerase thc hin s phin m chnh xc. 2.1.2.1- Cu trc ca promoter Promoter l trnh t nhn bit ca enzyme RNA-polymerase v l ni m enzyme RNA-polymerase gn vo xc nh v tr bt u phin m. Do vy nn promoter c nhng cu trc c hiu gip enzyme nhn bit chnh xc. Kho st nhiu promoter khc nhau ca cc gen, ngi ta nhn thy phn tm ca promoter c nhng trnh t chung ging nhau v gi l cc hp, v d E. Coli c hp TATAAT. Hp ny thng nm v tr khong 10,

tc l nm khong 10 nucleotide pha trc v tr khi u phin m hay trnh t TTGACA nm v tr 35, tc l khong 35 nucleotide trc v tr khi u phin m. vi khun c mt loi promoter v ch c mt loi RNApolymerase.
35 10
V tr khi u phin m ca RNA
T T GA C A
T AT AAT
CT G T T GA C A A T T AA T C A T C GAA C T A G T A T A A T A GT A C GC AA GT GA C A A C T G T T AA T T A GT A GC T T GA T C A T A T T A T C A T GC GT T C A
Hnh 2-1: Vng tm promoter ca operon Trp vi khun t bo eucaryote, vng iu khin thng ln hn t bo procaryote. Eucaryote c ba loi enzyme RNA-polymerase nn chng c ba loi promoter, mi loi ng vi mt loi enzyme chng d dng nhn bit v bm vo thc hin phin m. Promoter nhm I l v tr bm ca enzyme RNA-polymerase I, promoter nhm II v nhm III l v tr bm ca enzyme RNA-polymerase nhm 2 v nhm III. Mi loi promoter c nhng trnh t chung ging nhau, cc trnh t ny nh v nhng v tr xc nh, do , cc enzyme d dng nhn bit v thc hin phin m chnh xc. 2.1.2.2- Cu trc vng mang thng tin di truyn t bo sinh vt procaryote, cc gen c t chc theo dng operon, nghi l, mi mt gen mang m tng hp mt s chui polypeptide. Vng mang m tng hp mt polypeptide gi l mt cistron. Nh vy, gen t bo procaryote thuc loi polycistron. Cc cistron sp xp theo tng nhm, chung mt vng iu khin to thnh mt operon. Cc protein c m ha trong mt operon thng c lin quan cht ch vi nhau trong mt qu trnh chuyn ha sinh ha no trong t bo. Kiu t chc b my di truyn nh vy gip vi khun thch nghi nhanh vi nhng thay i ca iu kin ngoi cnh mi trng. Ton b vng mang thng tin di truyn c m ha cho cc polypeptide. Vng mang m di truyn ca sinh vt eucaryote c cu trc phc tp hn. Phn ln cc gen c cha cc on khng mang m (intron) nm xen k vi cc on mang m (exon). Ch c mt s t cc gen l khng c intron nh mt s gen m ha cho protein histon. nhiu gen, phn khng mang m (intron) c tng di ln hn tng di ca cc on mang m, nh gen

m ha cho albumin, conalbumin, ... S lng intron c mt trong cc gen cng khng ging nhau, v d nh gen m ha cho -globin ch c hai intron, trong khi , gen m ha cho colagen li c n 52 intron. Cc on intron s c ct b trong qu trnh phin m. im giao tip gia intron v exon c nhng du hiu ring bit, l cc cp baz GU v AG (...GU.....AG...). 2.1.2.3- Cu trc vng kt thc Vng kt thc nm u 5 ca si DNA lm khun (sense) - l u 3' ca gen (si antisense), bao gm nhng trnh t khng m ha cho cc axit amin. Vng ny thng c cc tn hiu dng phin m, gip enzyme RNApolymerase dng phin m ng v tr. Ngoi ra, trong vng ny cn c mt s trnh t c chc nng cha r rng. 2.2- S SAO M DNA (Replication) Mt trong nhng tnh cht cn bn ca DNA l kh nng t sao chp (replication) hay l t nhn i (self duplication). Ngha l t mt phn t DNA m sao chp to ra hai phn t DNA con ging y nh phn t DNA m ban u. S ti bn DNA l mt trong nhng tnh cht quan trng, nh m thng tin di truyn c truyn t th h ny sang th h khc. 2.2.1- L thuyt v sao m DNA theo khun 2.2.1.1- Gi thuyt ca Watson - Crick Sau khi xy dng m hnh cu trc ca phn t DNA, Watson v Crick thy rng, nu hai si DNA tch ri ra th mt si s lm khun tng hp mt si mi v do cc baz nit lun c xu hng bt cp vi nhau (A vi T v G vi C). Theo d on ca Watson v Crick, qu trnh tng hp DNA c th tm tt nh sau: - u tin lin kt hydr s b t ra to hai mch n lm khun. - Cc baz nit mi s bt cp b sung vi cc baz nit trn si lm khun theo qui tc A i din vi T v G i din vi C.

- Kt thc qu trnh sao m, t mt si DNA xon kp m s to hai si xon kp con. Mi si xon kp con c mt si mi tng hp v mt si s lm khun. Ngay sau khi m hnh c nu ra, nhiu th nghim chng minh c tin hnh xc nhn d on. 2.2.1.2- Th nghim chng minh ca Meselson - Stahl (1958) Th nghim ny nhm mc ch chng minh kiu tng hp theo khun ca DNA nh d on ca Watson v Crick. Th nghim c thc hin trn E. Coli. Vi khun c nui cy qua nhiu th h trn mi trng c cha nit ng v nng N
15 15
v nit thng N .
14
Nguyn tc ca th nghim l da trn kh nng phn bit t trng ca hai ng v N v N

14
c mt trong cc loi DNA khi li tm trn thang nng
CsCl2 . Qu trnh th nghim c tin hnh nh sau: - Th h u (gi l th h 0): vi khun c nui trn mi trng cha N
15 15
nh vy th h ny, cc nguyn t nit trong DNA ca vi khun s l N . Sau khi nui tin hnh tch DNA v em li tm trn thang nng CsCl2, nhn thy:
+
th h 0: ch cha 1 gii n c t trng cao. Nh vy tt c DNA ca th h cha m u cha 2 si vi nit nng.
- Th h 1: Chuyn vi khun th h 0 sang nui trn mi trng cha N . Thi gian v iu kin nui tung t nh th h 0. Sau khi tch DNA v em li tm nh th h 0, th thy:
+
14
th h 1 ch cha 1 gii n c t trng trung bnh. Nh vy mi phn t con th h 1 c 1 si cha nit nng ca b m v 1 si b sung mi tng hp c cu to t nit nh N , gi l si DNA lai.
14
- Th h 2: Tip tc chuyn vi khun ca th h 1 sang th h 2 v cng nui trn mi trng cha N . Qu trnh th nghim tin hnh tng t nh cc th h trn. Sau khi li tm, quan st thy c mt na s lng phn t DNA l phn t lai ging th h 1 v mt na s lng cn li l phn t DNA vi c hai si u mang N
14 14
(Hnh 2-2).

Th h 0: DNA nng 15 nui trn mi trng N
Th h 1: DNA lai 14 nui trn mi trng N
Th h 2: Nui 14 trn mi trng N
Hnh 2-2: M hnh th nghim ca Meselson-Stahl Th nghim trn l mt bng chng chng minh gi thuyt ca Watson v Crick l ng. Qu trnh tng hp DNA phi thc hin theo khun, hai mch ban u c tch ra, mi ci lm khun tng hp mch mi. Kt qu l, mi phn t con u mang mt mch c v mt mch mi. Kiu sao m nh vy gi l sao m bn bo tn (semi-conservative). 2.2.1.3- Th nghim chng minh ca Arthur v Kornberg Cng trong nm ny (1958), Arthur v Kornberg ti trng i hc Washington tinh sch mt loi enzyme t E. Coli, gi l DNA-polymerase. Enzyme ny xc tc tng hp mt si polynucleotide t cc nucleotide-3phosphat. Nguyn liu dng cho th nghim gm: - Bn loi nucleotide dng triphosphat: dATP, dTTP, dGTP, dCTP. - Enzyme DNA-polymerase chit t vi khun E. Coli. - Ion Mg l cofactor ca enzyme, to iu kin thun li cho hot ng ca enzyme.
+2

Tin trnh th nghim c th m t nh sau: Th nghim th nht c tin hnh vi y cc nguyn liu nh gii thiu trn, trong cc iu kin thun li cho s tng hp si DNA mi, nhng khng c si lm khun. Th nghim th hai cng c tin hnh tng t nh th nghim 1 nhng c mt si DNA lm khun. Si lm khun c thu nhn bng cch lm bin tnh v tch mch, sau a vo th nghim. H thy rng, th nghim th nht, enzyme DNA-polymerase khng th tng hp si DNA t cc nguyn liu khi khng c mt ca si khun, vic tng hp si DNA mi ch xy ra th nghim th hai, khi c mt DNA khun. iu khng nh rng gi thuyt ca Watson v Crick l ng. DNA c tng hp ch khi c mt si lm khun. 2.2.2- Qu trnh sao m 2.2.2.1- Cc enzyme v protein c hiu tham gia sao m y l mt qu trnh phc tp, thc hin sao m cn thit phi c mt ca cc enzyme, cc protein c hiu v cc iu kin cn thit sau y: 1,- Lin kt hydro gia 2 mch b sung ca si DNA m phi b ph v v tch ri tng bc lm 2 mch lm khun. 2,- Phi c on mi (primer), tc on RNA mch n ngn bt cp vi mch n DNA khun t v tr bt u ca mi on sao chp. 3,- C 4 loi nucleotide dng triphosphat (dATP, dTTP, dGTP, dCTP) bt cp b sung vi cc nucleotide mch khun. 4,- Mch mi c tng hp theo hng 5P 3OH. 5,- Mi bc c iu khin bi enzyme c hiu v c thc hin mt cch nhanh chng, chnh xc. 6,- C mt ca ion Mg
+2
lm cofactor ca enzyme.
7,- Trong qu trnh sao chp, ngoi cc enzyme c hiu cn c cc protein c hiu.

Cc enzyme v protein c hiu tham gia sao chp gm c: - Enzyme topoisomerase, lm nhim v tho xon v hai pha trn si DNA kp. - Enzyme helicase, s dng nng lng ATP lm t lin kt hydr gia cc baz nit ca hai mch khun. - DNA-polymerase I, -II, -III lm nhim v tng hp si mi v sa sai. - RNA-polymerase, lm nhim v tng hp on mi. - Enzyme primase, lm nhim v gn mi. - Enzyme ligase, lm nhim v ni hai nucleotide ng cnh nhau bng lin kt phosphodiester to mch polynucleotide. - Enzyme ribonuclease (RNase) lm nhim v ct b mi sau khi tng hp xong mch mi.
g g t a C C A T t A g C t a C C a t g g A t C Ag t A t T C g a g c
DNA con
A
A
A
C
G
t c
g
G
C
T a
DNA m
g c C g t a
g c A t C g C g t a A t t a
Hnh 2-3: Sao chp theo quy lut b sung Cc protein tham gia sao m gm c: - Protein B, nhn bit im khi u (origin) sao chp trn si DNA kp. - Protein SSB (Single Strand Buiding), lm nhim v gi hai mch ca si DNA khng cho chp vo nhau, to iu kin thun li cho qu trnh sao chp.

2.2.2.2- Qu trnh sao m 1,- Nhn bit im khi s v tho xon DNA: Qu trnh sao m c nghin cu kh k t bo vi khun E. Coli. S sao m bt u khi protein B c hiu nhn bit im khi s sao m (replication origine) v gn vo trnh t c hiu . Tip theo, enzyme topoisomerase thc hin tho xon phn t DNA t im khi s. Enzyme helicase s dng nng lng ATP ct t cc lin kt hydro gia cc baz bt cp ca hai mch phn t, tch hai mch to thnh chc ba hnh ch Y, gi l chc ba sao m (replication fork). C nhiu loi enzyme helicase: c loi gn trn mch, di chuyn v ct lin kt hydro theo chiu t u 3' n u 5'; c loi gn ln mch, di chuyn v ct lin kt hydro theo chiu 5' 3'. Sau khi tch ri, hai mch n s c protein lm cng mch SSB gi khng cho chp li, lm cho trng thi m xon c bn vng. Mi phn t protein SSB bm vo 8 nucleotide trn mch n, mi chc ba sao m c khong 250 phn t protein SSB hot ng. Mch khun c s dng n u, cc phn t protein SSB s c gii phng n . 2,- Tng hp mi: Enzyme DNA-polymerase ch c kh nng tng hp si DNA mi bng cch ni di u 3'OH t do ca mt on mi bt cp sn trn khun theo chiu 3'OH n u 5'P. Mi l mt on RNA nh chng 10 nucleotide, c tng hp t khun ca si DNA. Phc hp enzyme primaseRNA-polymerase bm vo mch n ca chc sao m tng hp on RNA mi to u 3'OH t do ca on mi. Enzyme DNA-polymerase III xc tc tng hp mch b sung t u 3'OH t do ca mi, ko di mch. S tng hp cc mch n DNA mi, v vy, ch i theo mt chiu xc nh t u 5' n u 3' (5'3'). 3,- S tng hp mch DNA mi xy ra mt cch lin tc trn mch khun c chiu 3' 5' v gin on trn mch khun c chiu 5' 3': Trn mch khun c chiu 3'5', mch mi c tng hp theo chiu 5'3' mt cch lin tc, cng hng tho xon ca phn t DNA. on mi s c tng hp t im khi s sao m. Khi xut hin u 3'OH t do ca on mi th enzyme DNA-polymerase III gn vo v tng hp ngay mch b

sung theo chiu 5'3' mt cch lin tc cho n im kt thc sao m. Mch ny c tng hp nhanh hn nn ngi ta thng gi l mch nhanh (leading strand). Nh vy, tng hp c si DNA mi b sung cho si lm khun ny, enzyme DNA-polymerase ch cn c mt on mi (Hnh 24).
3
Chc ba sao m
5
Mch chm
5 3 5 3
Hng sao m
Hnh 2-4: S biu din cch sao chp gin on v lin tc trn 2 si DNA khun Trn mch khun c chiu t u 5'3', vic tng hp mch mi phc tp hn, do qui lut tng hp si DNA mi b sung lun thc hin theo chiu t u 5'3'. Mch mi b sung cho mch khun ny c tng hp di dng tng on ngn gi l okazaki. Mi mt okazaki c mt on mi. vi khun, di mt okazaki khong 1.000 n 2.000 nucleotide, cn t bo eucaryote, s lng nucleotide trong mt okazaki ngn hn so vi vi khun. Hng di chuyn tng hp si mi ca DNA-polymerase III ngc vi hng tho xon ca phn t DNA m. Si con th hai ny c tng hp mt cch chm hn nn thng gi l mch chm hay mch sau (lagging strand). Cc on RNA mi sau s b enzyme ribonuclease phn hu. Cc l trng xut hin sau khi cc on mi mt i s c lp y nh hot ng ca enzyme DNA-polymerase I. Cui cng, enzyme DNA-lygase ni cc lin kt phosphodiester gia cc on, to nn mch DNA con hon chnh. Ngoi chc nng tng hp si DNA mi theo chiu t u 5'3', enzyme DNA-polymerase III cn c kh nng sa sai nh hot tnh exonuclease. Trn ng di chuyn tng hp, nu n gp ch m

nucleotide mi bt cp sai, n s li li ct b nucleotide sai v lp nucleotide ng vo. Kt qu, sau qu trnh sao m, hai phn t DNA con c hnh thnh c cu to ging y nh phn t DNA m ban u, m bo thng tin di truyn c truyn t th h ny qua th h khc mt cch chnh xc. 2.2.3- Sao m DNA si kp dng vng ca t bo procaryote 2.2.3.1- Sao m theo kiu hnh mt
Hng sao m im khi s sao m
Hng tho xon
Hng tho xon
Sao m thc hin theo 1 hng
Sao m thc hin theo c 2 hng
Hnh 2-5: S tng hp DNA dng vng vi khun DNA ca t bo procaryote thng c dng xon kp hnh trn. Qu trnh tng hp cng c thc hin t mt im khi u (replication origine) gi l im xut pht v trin khai ra c hai pha chc ba sao m v lan dn v hai pha, cui cng to ra hai phn t DNA lai. C trng hp s tng hp ch xy ra v mt pha ca im khi u. Khi DNA dng vng trn ang sao chp, quan st thy c dng hnh con mt (eye replication). Mt n v sao m thng nht (t mt im xut pht) gi l replicon. B gen ca cc sinh vt procaryote ch c mt replicon. Tc tng hp DNA ca E. Coli c th t 50.000 nucleotide/pht, chu k sao m ko di khong 20 pht (Hnh 2-5).

2.2.3.2- Sao m kiu hnh trn xoay
Ct lin kt phosphodiester 5
Si mi c tng hp t u 3 5 mt cch lin tc
SSB
5
Replisome
Si khun c chiu 5 3 c tng hp mt cch gin on
Okazaki trc
on mi Okazaki sau
Hnh 2-6: Sao m theo kiu hnh trn xoay Ngoi kiu sao m c dng hnh con mt nh trn, mt s vi khun v virus cn c kiu tng hp khc gi l sao m hnh trn xoay. Qu trnh tng hp bt u bng s ct lin kt phosphodiester ti mt im xc nh trn mt si DNA trn kp to ra hai u mt, mt u kt thc bng nhm 3'OH v u kia l u 5'phosphat. S tng hp mt mch mi mt cch

lin tc bng cch ko di mch t u 3'OH ng thi dch chuyn theo dng xoay trn, u 5'P c xoay ra ngoi v mt mch mi khc c tng hp gin on theo okazaki, sau khi kt thc, hai phn t DNA con c hnh thnh (Hnh 2-6). 2.2.4- Sao m t bo eucaryote
t bo eucaryote c nhiu nhim sc th, a dng, mi nhim sc th
l mt phn t DNA nm lin kt vi protein, nn qu trnh sao m phc tp. Nhiu im sao m xy ra ng thi, hay ni cch khc l trong cng mt thi im c nhiu n v sao m (replicon). V d nh nm men bnh m S. Cerevisiae c 500 replicon.
5
5 3
3
Qu trnh tng hp DNA t bo eucaryote phc tp hn v tc chm hn (3.000 nucleotide/pht). c im quan trng trong qu trnh sao m t bo eucaryote l c nhiu n v sao m xy ra, ng thi, trn mt phn t DNA v t bo c c ch kim sot nghim ngt qu trnh ny. im no sao qua mt ln ri th khng lp li trc khi ton b phn t DNA c ti to hon ton. Sau khi sao m, cc DNA con c cu to ging y nh DNA m ban u c phn chia u n v mi t bo con trong qu trnh phn bo. 2.2.5- Sa sai DNA trong t bo 2.2.4.1- Sa sai trong sao m Sa cha nhng sai st trn DNA nhm khi phc li cu trc ban u l mt c tnh ca mi t bo sng. Trong qu trnh sao m, cc baz nitt cng c th bt cp sai. S bt cp sai thng xy ra l A bt cp sai vi C (A...C) v G bt cp sai vi T (G...T). S d c s bt cp sai nh vy l do trng thi tn ti ca cc baz c nhng thay i, dn n s nhn bit sai.

Nh c ch tng hp DNA lun lun khi ng t u 5 3, nn vic sao m c kim sot v sa cha mt cch chnh xc. Cc enzyme DNA-polymerase I v III va lm chc nng polymer ha va c hot tnh exonuclease theo hng 5 3 v 3 5. Nu trn ng di chuyn bt gp mt nucleotide lp sai, chng s li li ct b v lp nucleotide ng vo. Khi h thng kim tra pht hin c sai st, cc enzyme endonuclease s ct b on sai, sau , enzyme DNA-polymerase I s tng hp li cho ng v enzyme DNA-ligase s ni li phc hi trng thi bnh thng. Khi tng hp nhn to phn t DNA (invitro) ngi ta c tnh sai st l 10
5 6 5
, ngha
l, trong 10 nucleotide c tng hp th c 1 nucleotide sai. V d DNA trong t bo E. Coli c 3x10 nucleotide, nh vy c sau mi ln sao m th c 30 nucleotide sai, hay cc nhim sc th ngi c 3x10 nucleotide, nh vy c sau mi ln sao m th c 30.000 nucleotide sai. Nu vi tn xut sai ny th s lng t bin s rt ln. Qua thc t nghin cu cho thy tn xut t bin trong cc qun th sinh vt l nh hn rt nhiu. Bng cch nh gi tn s t bin xut hin trong cc qun th, ngi ta c tnh s sai st trong sao m nm trong khong 10 . Tuy nhin, trong thc t, t l sai st cn thp hn nhiu, vo khong 10 n 10 , iu ny chng t s tn ti mt h thng kim tra v sa sai rt hiu qu ca t bo. 2.2.4.2- Sa sai sau khi sao m Mi trng sng v c th sng c mi quan h mt thit vi nhau. Cc bin ng ca mi trng sng lun tc ng n cc sinh vt. Nhng thay i ca mi trng bn ngoi c tc ng trc tip n b my di truyn, lm bin i n. 1,- Cc tc nhn c th lm thng tn DNA trong qu trnh tn ti: - Cc tia v tr v cc tia phng x c nng lng cao c th lm bin i cc baz nit nh gn thm cc nhm chc khc vo mch vng hay lm t vng, lm t cc lin kt hydro gia hai mch hay lm ct mch ca DNA,... - Cc tia cc tm trong nh sng mt tri thng gy nn s dimer ha cc baz thymine, hnh thnh lin kt gia hai baz thymine nm k nhau, lm chng mt kh nng lin kt vi baz adenine ca mch b sung.
10 11 9 9

- Tc ng ca cc thnh phn trong ni bo. S tn thng phn t DNA c th gy ra do qu trnh trao i cht bt bnh thng gy ra cc cht c hi v cc gc t do bt li, hay do hot ng ca cc enzyme khng ng b lm tn ng nhng sn phm trung gian bt li. Ngoi ra, cn c nhiu tc nhn ha hc khc ca mi trng bn ngoi cng gy nn s bin i c th xy ra trn DNA. 2,- Mt s kh nng gy bin i trn phn t DNA: - Gy mch hay t mch: Do si DNA rt mnh, bn thn n li xon cun nhiu ln nn rt d b gy hay t mch do tc ng bn ngoi hay khi tho xon. - Mt cc baz b sung, ngha l lm cho baz tng ng khng c cp (nh mt baz purine). - Bin baz nit ny thnh baz khc, gy nn s bt cp sai, nh: Khi mt nhm amin, cytosine s bin n thnh uracil hoc 5-metyl-cytosine-desamin b nhn nhm l thymine. - Cc baz nit c th tn ti di 2 dng ceton v enol nn dn dn bt cp sai. - Gn thm nhm CH3 , C2H5: Khi gn thm nhm ankyl s lm thay i tnh cht ca cc baz nit dn n bt cp sai. 3,- C ch phng nga v sa sai: bo v v thch nghi vi nhng bin i, t bo c c ch phng nga v sa sai. Cc c ch phng nga ca t bo nh h thng enzyme kh c, loi b cc c t, h thng iu ho cn bng cn thit cho t bo, h thng enzyme tham gia sa sai. V d sau cho thy h thng sa sai v phng nga ca t bo: - Enzyme Superoxide Dismutase (SOD) lm nhim v gim c: Enzyme SOD c pht hin nm 1968, c m s EC.1.15.1.1 , c mt trong tt c cc t bo c chuyn ha oxy. Chng xc tc phn ng phn hu gc superoxyt O2 trong t bo theo s phn ng sau:

2O2
+ 2H +
H2O2 + O2
H2O2 s c enzyme catalase hoc peroxydase chuyn ha. Do , SOD cng vi catalase c xem l nhn t quan trng c th gii c, bo v v chng lo ha cho t bo. Gc O2 c sinh ra lin tc v cng b phn hu khng ngng bi hot ng ca SOD, do , khi SOD c hot cng cao th nng O2 cng thp. - H thng sa sai trong t bo rt a dng v c hiu qu vi s tham gia ca cc enzyme, nh photolyase, DNA-metyl-transfersase, h thng enzyme m ha trong h thng gen SOS. vi khun E.Coli, do tc ng ca tia t ngoi lm xut hin cc thymine dimer (T=T) trn DNA. Khi c nh sng, enzyme photolyase s c hot ha v ct b lin kt T=T, chuyn v trng thi bnh thng v n nh. H thng enzyme nuclease c th ct b ch sai hng bng nhiu cch v tng hp li cho ng. Enzyme DNA-metyl-transferase c kh nng loi b gc metyl trong trng hp cc baz b metyl ha. Tm li, t bo c c ch kim tra v sa nhng sai st nu c. Ngoi ra, t bo cn c h thng SOS (cp cu): H thng ny hot ng khi t bo b tc ng mnh bi cc tc nhn gy t bin, to nhiu sai hng trn DNA. Trong trng hp cp bch c nhiu sai hng cn cp cu, cc gen SOS c m ra. Nu sa sai khng kp, t bo phi chp nhn hoc b t bin hoc cht. Trong t bo cn c h thng iu ho cn bng axit-baz, to iu kin thun li cho hot ng trao i cht lm gim kh nng to cc cht c hi. sinh vt a bo, vic kh c v loi b nhiu ha cht gy c c m bo bi gan v thn. 2.3- S PHIN M (Transcription) Qu trnh tng hp phn t RNA t khun DNA c gi l qu trnh phin m (transcription). Nhng thng tin di truyn c trong phn t DNA c s dng tng hp protein. Tuy nhin, phn t DNA khng phi l cht lm khun tng hp protein, bi v, cc phn t DNA tp trung ch yu trong nhim sc th,


phn b trong nhn t bo, cn qu trnh tng hp protein li din ra trong t bo cht. Nh vy, phi c mt cht trung gian lm nhim v chuyn thng tin di truyn t DNA trong nhn ra t bo cht v lm khun cho qu trnh tng hp protein. Nm 1957, sau khi khm ph ra cu trc phn t DNA, Francis Crick nu tng v mt cht trung gian truyn thng tin di truyn cho qu trnh tng hp protein, v d on "ngi phin dch" trung gian l RNA. D on ny c khng nh bng cc nghin cu chng minh RNA c cu to ha hc tng t nh DNA v c tng hp trn khun ca DNA. Nm 1960, c 3 nhm nghin cu c lp M chit tch c enzyme RNA-polymerase, enzyme ny c kh nng tng hp phn t RNA t khun DNA. Benjamin Hall v Sol Spiegelman (1961) tch v tinh sch RNA v DNA t thc khun th T2 . Ly DNA ca T2 lm bin tnh tch thnh 2 si n. Khi cho cc si n DNA bt cp vi si n RNA th chng to thnh dng phn t lai DNA/RNA. Tuy nhin, khi h ly DNA ca cc c th khc trn vi RNA ca T2 th chng khng bt cp, ngha l khng to phn t lai. iu chng t rng, RNA ca T2 c tng hp trn khun DNA ca n theo c ch b sung nn chng c th bt cp v to thnh phn t lai. Nhng bng chng khc nh RNA tp trung ch yu t bo cht nhng chng cng c mt trong nhn t bo. nhng loi t bo m s tng hp protein xy ra mnh m th hm lng RNA tp trung nhiu hn so vi nhng t bo t tng hp protein. Nh vy, RNA chnh l cht trung gian truyn thng tin di truyn tng hp protein. Mi quan h gia DNA, RNA v protein c th hin nh sau: DNA
Sao m Phin m (Transcription)
mRNA
Dch m (Translation)
protein
V sau, ngi ta cn pht hin qu trnh phin m ngc, ngha l, phn t DNA c tng hp trn khun ca phn t RNA. Qu trnh phin m ngc c enzyme phin m ngc (reverse transcriptase) xc tc.

Enzyme ny c pht hin ln u tin khi nghin cu s sao chp b gen ca virus gy ung th (retrovirus). u tin, enzyme phin m ngc s tng hp si DNA n (cDNA) trn khun ca phn t RNA, sau , enzyme DNA-polymerase s tng hp mch b sung to thnh si DNA kp (cDNA kp). Qu trnh c th biu din qua s sau: RNA
(si n)
E phin m ngc
cDNA
(si n)
ADNpolymerase
cDNA
(si kp)
Phin m
mRNA
Dch m
protein
2.3.1- c im chung ca qu trnh phin m S phin m c thc hin theo mt s nguyn tc chung sau: - Sn phm phin m l mt si polyribonucleotide mch n (RNA). - thc hin phin m, phn t DNA phi dn xon cc b. i vi mi gen, ch mt trong hai si DNA c chn lm khun tng hp phn t RNA. Enzyme RNA-polymerase s quyt nh vic chn si lm khun v xc tc qu trnh tng hp phn t RNA. - Phn t RNA mi c tng hp theo chiu t u 5'phosphat n u 3'OH. - S phin m c thc hin khi c mt ca cc enzyme v 4 loi nucleotide l GTP, CTP, UTP v ATP. - Phn t RNA c tng hp theo c ch b sung tng t nh s sao m DNA nhng i din vi A trn si DNA s l U trn RNA. - Khng c c ch sa sai i km vi qu trnh phin m, do vy, nhng sai st s dn n s sai khc trong biu hin gen. Tuy nhin, s sai khc trong qu trnh phin m khng nh hng g n vic truyn t thng tin di truyn cho th h sau.

2.3.2- Phin m tng hp mRNA procaryote 2.3.2.1- Enzyme RNA-polymerase procaryote cc sinh vt procaryote ch c mt loi enzyme RNA-polymerase, n chu trch nhim xc tc qu trnh tng hp c ba loi RNA l mRNA, tRNA v rRNA. RNA-polymerase ca vi khun E. Coli c nghin cu k nht. Enzyme ny c cu trc bc 4 rt phc tp, bao gm hai hp phn chnh l enzyme li v nhn t . Nhn t c th tch ra khi enzyme li, c trng lng phn t l 70.000dalton (1dalton=1,66x10 g=1/12 khi lng nguyn t carbon). Enzyme li c cu to t 5 chui polypeptide: hai chui , mt chui , mt chui ' v mt chui . Ngoi ra, enzyme li cn c nhn t phin m l v nus A. Cc nhn t phin m ny d dng tch ra khi enzyme li, nn trong qu trnh tch v tinh sch enzyme thng khng tm thy. Bng 2-1: Cc tiu n v RNA-polymerase ca E.Coli Tiu n v S lng ' (beta) (beta) (alpha) (omega) (sigma) (rho) Nus A 1 1 2 1 1 6 1 Trng lng phn t (dalton) 160.000 150.000 42.000 11.000 70.000 46.000 70.000 Chc nng - lin kt vi DNA khun - ko di mch RNA - cha r - cha r - nhn bit promoter - kt thc phin m - ko di v kt thc
-24
T liu da theo S.C. Rastogi Gia cc si polypeptide trong enzyme li khng c lin kt ng ha tr bn vng m ch hnh thnh cc lin kt th cp. Trng lng phn t ca cc chui polypeptide c biu din Bng 2-1.

Chc nng ca chui ' l lin kt vi si DNA khun, cn chui c tc dng xc tc hnh thnh lin kt phosphodiester. Vai tr ca hai chui hin cha bit r. Nhn t ng vai tr quan trng trong vic tng hp RNA, n gip cho enzyme li c th khi u qu trnh phin m ti mt im c th. Nhiu th nghim (in vitro) cho thy, khi thiu nhn t , enzyme li s khi u phin m mt cch ty tin, khng ng v tr quy nh. E. Coli, ngi ta tm thy 3 loi nhn t , mi loi c trng lng phn t ring. RNA-polymerase pht hin ra promoter, sau gn ln phn t DNA, m xon cc b v thc hin qu trnh tng hp phn t RNA. S c mt ca Mg
+2
l cn thit cho hot ng xc tc ca enzyme RNA-polymerase.
'
Hnh 2-7: Cu trc RNA-polymerase procaryote 2.3.2.2- Phin m tng hp mRNA procaryote c im ca mRNA procaryote l mi phn t mang d liu thng tin cho s tng hp nhiu si polypeptide ng thi (polycistronic mRNA). Phin m tng hp mRNA procaryote c nghin cu kh k vi khun E. Coli. Qu trnh ny c th chia lm 3 giai on: khi ng, ko di v kt thc. 1,- Giai on khi ng: Tiu n v ca enzyme RNA-polymerase nhn bit v gn enzyme vo promoter khi ng qu trnh phin m. Promoter l on phn t

DNA c cu trc c bit, gip enzyme RNA- polymerase nhn bit v tr bt u ca s phin m. Enzyme RNA-polymerase gn vo trnh t 35 ca ptomoter, trt dc theo phn t DNA n trnh t 10 th m xon, lm l si lm khun, on m xon c di khong 1217 nucleotide, qu trnh phin m bt u ti v tr xc nh.
35 TTGACA 10 TA TA A T AAT GT GT GGAAT 5 CCCC AGGC TTGACA C T T T A T GC T T CC GGC T C G TAT 3 GGGGT CC G AAC T GT GAAA T A C GAA GGCC GA GC A TA P P P AA
T AC C A C A CC T T A T GT GAGC 3 ACAC T CG
im khi u phin m
Hnh 2-8: Khi u phin m promoter UV5 operon lactose Sau khi tng hp c on RNA ngn khong 810 nucleotide th nhn t tch khi enzyme li v si khun DNA, kt thc giai on m u. Sau , nhn t c th kt hp vi mt enzyme li khc khi u mt s phin m mi. 2,- Giai on ko di: Sau khi nhn t tch khi phc hp, mt nhn t ko di l protein Nus A gn vo, enzyme DNA-polymerase tip tc chuyn dch dc theo gen, lm gin xon si DNA v thc hin qu trnh polymer ha, ko di si RNA mi. Si mRNA c tng hp theo nguyn tc b sung: A i din vi U, T i din vi A, G i din vi C v C i din vi G, nh sau: Si khun DNA: Si RNA mi: A U T A G C C G
Khi enzyme RNA-polymerase trt dc theo gen n u th s tho xon v tng hp mi c thc hin n , cn on gen sau khi tho xon, phn t RNA-polymerase trt qua ri th c xon tr li cu trc ban u (Hnh 2-9).
Gen khi u
im kt thc
DNA
bm vo RNA-polymerase
RNApolymerase
NusA ri khi E
E
gip E nhn bit gen khi ri khi RNA v RNApolymerase
PPP
u bt u phin m Nucleotide triphosphat
bm vo RNA v polymerase tch ra ngay sau khi phin m bt u
NusA bm vo
PPP
PPP
RNA c phin m
Nucleotide triphosphat
Si RNA phin m di thm ra
RNA-polymerase Nhn t rho ()
Nhn t sigma () Protein NusA
Hnh 2-9: S biu din qu trnh tng hp RNA 3,- Giai on kt thc: Trn si DNA khun c cha on m kt thc, c chc nng lm dng qu trnh phin m im qui nh. Ti im kt thc, enzyme RNApolymerase dng qu trnh ni cc nucleotide, gii phng phn t RNA mi tng hp ra khi phc h enzyme v DNA khun, ng thi, enzyme cng tch khi si DNA c th n vi mt trnh t khi ng mi.

C hai kiu kt thc phin m: - Th nht: Enzyme RNA-polymerase tip nhn tc nhn kt thc . - Th hai: Kt thc xy ra khng cn s c mt ca tc nhn . Khi nghin cu v cch kt thc phin m theo kiu th hai vi khun v phage, ngi ta nhn thy c hai im c trng v cch sp xp cc baz nit on cui trn si lm khun nh sau: - C 2 trnh t i xng b sung pha trc im kt thc. - Mt on poly A nm ngay trc im kt thc (Hnh 2-10). Nh cu trc c bit nh vy nn sau khi hai trnh t i xng b sung c hnh thnh trn si RNA, chng c th bt cp vi nhau, to thnh cu trc c dng "kp tc", ngn cn RNA-polymerase phin m tip tc, ng thi, c tc dng ko si RNA mi c tng hp ra khi phc h enzyme v si khun. Qu trnh tng hp s dng li on poly-U.
5 CC CA G C C C G C C T A A T GAG C G G G C T T TTT T TT T GA A CAAAA 3 Si khun Dna : 3_ GGGT C G GG C G GA T TA C TC G C CC G A AAAAAAAA C TT GTT TT _ 5
RnA: 5_ CCCAG CCCGC C UA A UGAGCGGGC UUUUUUUU _ OH 3

A U C C G C Dng kp tc: C C G 5 C C C A A U G A G C G G G C U U U U U U U U OH3
Hnh 2-10: S biu din cch hnh thnh cu trc dng "kp tc" Ti cc v tr kt thc cn s c mt ca protein kt thc phin m , cu trc ca on DNA khun thng thy thiu on poly-A trc im kt thc v khng phi lc no cng c cc trnh t i xng b sung c th hnh thnh dng "kp tc" nu trn.

Hin nay, chng ta cn cha bit r, bng cch no m nhn t tc ng ln qu trnh kt thc phin m. C th n s dng nng lng t phn t ATP thc hin s tc ng ny. Mt c im ni bt t bo sinh vt procaryote l cc phn t DNA nm trong t bo cht, nn qu trnh phin m v dch m c tin hnh ng thi. 2.3.3- Phin m tng hp mRNA eucaryote S phin m eucaryote xy ra phc tp hn nhiu so vi procaryote. c im ca mRNA eucaryote l mi phn t mang d liu thng tin cho s tng hp mt polypeptide. Qu trnh phin m c thc hin trong nhn t bo, cn s dch m xy ra t bo cht, hai qu trnh ny xy ra hai v tr khc nhau. 2.3.3.1- Enzyme RNA-polymerase eucaryote t bo eucaryote c 3 loi enzyme RNA-polymerase, mi loi m nhn mt chc nng ring bit, RNA-polymerase I chu trch nhim tng hp rRNA; RNA-polymerase II chu trch nhim tng hp mRNA v RNApolymerase III chu trch nhim tng hp cc loi RNA c kch thc nh nh tRNA, RNA 5S ribosome v cc phn t RNA nh khc. Cu trc ca cc RNA-polymerase sinh vt eucaryote rt phc tp, c trng lng phn t ln v cng c hnh thnh t nhiu tiu n v nh enzyme RNA-polymerase sinh vt procaryote. S lng tiu n v cu to thng khong 10. Mi loi RNA-polymerase chu trch nhim chnh tng hp mt loi RNA, do vy, cc RNA-polymerase khc nhau s c kh nng nhn bit promoter ca mnh khi u phin m ng v tr. Nhiu kt qu nghin cu cho thy, eucaryote, trnh t on DNA promoter di hn so vi t bo procaryote, c bit l i khi cn c s hin din ca nhm trnh t khuych i (enhancer), c tc dng lm tng biu hin gen. Ngoi 3 loi RNA-polymerase ch yu k trn, trong t bo sinh vt eucaryote cn c cc RNA-polymerase ca ty th v ca lp th. Cc enzyme ny chu trch nhim phin m cc gen nm trn DNA ty th v lp th.

2.3.3.2- Qu trnh phin m eucaryote DNA ca t bo eucaryote tp trung ch yu trong cc nhim sc th trong nhn, c phn t lng ln v cha nhiu gen. Phn ln cc gen, phn mang m tng hp protein, c s an xen gia cc on mang m (exon) v cc on khng mang m (intron). Do vy, qu trnh phin m xy ra phc tp hn v thng bao gm hai qu trnh chnh: - Qu trnh phin m to nn phn t mRNA u tin, gi l phn t tin mRNA. - Qu trnh bin i phn t tin mRNA thnh phn t mRNA trng thnh. Qu trnh phin m to nn phn t tin mRNA cng c thc hin theo cc giai on tng t nh qu trmh phin m t bo procaryote: Tc l, enzyme RNA-polymerase cng vi tc nhn khi ng phin m nhn bit promoter v khi ng phin m v tr chnh xc, tip theo l qu trnh ko di mch mRNA v kt thc phin m ti v tr xc nh. Enzyme RNApolymerase II s chu trch nhim phin m tng hp phn t mRNA. Phn t tin mRNA tri qua mt s bin i mi tr thnh phn t mRNA trng thnh. Qu trnh bin i bao gm: gn m, hnh thnh ui polyA v loi b cc intron, ni cc exon. 1,- Gn m (capping): Phn t mRNA c tng hp theo hng t u 5P n u 3OH. Sau khi phn t tin mRNA hnh thnh c mt on th u 5P c gn thm mt nucleotide l 7methylguanosine (Hnh 2-11). Vi chiu ngc li to lin kt phosphodiester gia hai cacbon th 5 ca hai nucleotide. To mch lin kt 55 thng c gi l m. Sau khi c gn m th u 5 ca phn t mRNA cng s c nhm 3OH t do gc ng ging nh u 3. Tuy vy, nh nhm metyl (CH3) v tr nit th 7 ca baz guanosine c th phn bit c u 5. Nh vy, sau khi gn m, u 5 khng cn nhm phosphat t do na. M gip cho ribosome nhn bit v gn vo u 5 ca mRNA, khi u dch m ng v tr qui nh, ng thi, m bo v mRNA khi b cc enzyme nuclease phn hu.

O N N h2N N N O O O 5 5 Ch2 O P O P O P O Ch2 OH O 3 O O 3 2 O Ch3 Baz O OH OH ch3
Baz O
OH HO
HO P O Ch2
2 O (Ch3 hoc H)
m GpppNm (Nm)N 5
aaaaaa (n) a aa oh 3
Hnh 2-11: Cu to "m" chp u 5' ca mRNA 2,- Hnh thnh ui poly-A u 3OH: Ngi ta pht hin rng, trong phn ln cc phn t mRNA ca nm men v t bo eucaryote, u 3 c cha mt trnh t di khong 200 nucleotide vi cc baz adenine (A) gi l ui poly-A. ui poly-A l im c trng ca cc phn t mRNA eucaryote. cc loi RNA khc nh rRNA, tRNA khng c ui poly-A. ui poly-A c gn vo sau khi phn t tin mRNA c tng hp xong. Enzyme chu trch nhim gn ui poly-A vo tin mRNA l poly-A-polymerase. Qu trnh hnh thnh ui poly-A c tin hnh theo m t Hnh 212. u 3'OH ca phn t tin mRNA c cha mt trnh t nhn bit l AAUAAA. Mt enzyme endonuclease c hiu nhn bit v ct si tin mRNA mt v tr khong t 11 n 30 nucleotide sau trnh t nhn bit v pha u 3'OH, tip sau , enzyme poly-A-polymerase s xc tc gn cc nucleotide A vo u 3'OH to thnh ui poly-A. di ui poly-A ca cc mRNA thay i tu theo loi v giai on pht trin ca t bo. Khi mRNA di chuyn t nhn ra t bo cht cng nh trong qu trnh tn ti, ui poly-A b thoi ha ngn dn. ui poly-A cng di, thi gian tn ti ca mRNA trong t bo cng lu. Trong mt s t trng hp, mRNA

khng c ui poly-A nh cc mRNA phin m t gen m ha histon khng c ui poly-A.
AA T AAA T T AT T T Phin m AA U AAA 5 Enzym endonuclease nhn bit trnh t AAUAAA v ct m-RNA khong 11 n 30 nucleotide AA U AAA 5 Enzyme poly-A polymerase gn ui poly-A AA U AAA 5 3 mRnA
Ct
AAAAAAAAA 3
ui poly-A (khong 200 nucleotide)
Hnh 2-12: S v s hnh thnh ui poly-A 3,- Ct b intron v ni cc exon (splicing): Cc gen sinh vt eucaryote thng rt di. Phn ln c cha cc on khng mang m cho protein (intron) xen ln vi on mang m (exon). Nhiu gen c tng di ca cc intron ln hn tng di ca cc exon. V d nh gen m ha cho -globulin c 2 intron: Mt intron nh c di 116 cp baz v mt intron ln c di l 646 cp baz, thm vo , u 5 c 52 cp baz v u 3 c 110 cp baz khng mang m cho protein. Tng di ca cc on khng mang m ln hn tng di ca cc on mang m (Hnh 2-13). Tng t nh vy, gen m ha cho ovalbumin cng c tng di ca cc on intron ln hn tng di ca cc on exon cng li.

Intron Intron
gen
mRNA 0
ui
AAAA (150) 1,6 2,0Kb
0,4
0,8
1,2
Hnh 2-13: So snh di ca mRNA ca -globulin ca chut vi di ca gen m ha i vi cc gen c cha intron th sau khi hnh thnh bn phin m u tin phi tri qua giai on ct b intron v ni cc exon to thnh phn t RNA trng thnh. Qu trnh ny xy ra theo hai bc chnh: Cc endonuclease thc hin vt ct ranh gii gia intron v exon, tip theo l s ni cc exon li v loi b intron. Trong qu trnh loi b intron v ni cc exon c s tham gia ca cc phn t ghp ni (spliceosome) l phc hp gia RNA c phn t lng nh giu uracil c mt trong nhn t bo vi mt s protein chuyn bit trong nhn. Sau khi cc intron c loi b, cc exon c ni li, phn t mRNA trng thnh lc ny c m chp u 5, ui poly-A u 3 v cc trnh t mang m cho protein. mRNA trng thnh s i qua l mng nhn vo t bo cht kt hp vi ribosome thc hin qu trnh tng hp protein. Tuy nhin, khng phi tt c cc gen trong t bo sinh vt eucaryote u c cha intron. C mt s gen khng c intron, v d nh phn ln cc gen m ha cho cc protein histon. Trong trng hp ny, qu trnh phin m to mRNA s khng c giai on splicing. 2.3.4- Phin m tng hp tRNA Trong b gen ca t bo procaryote cng nh eucaryote c cc gen m ha cho tRNA. vi khun, thng thng mt gen c th m ha cho mt s tRNA khc nhau. V d E. Coli, mt gen c th m ha cho 7 loi tRNA khc nhau. Tuy vy, i lc ngi ta cng tm thy c nhng gen ch m ha cho mt tRNA.

Qu trnh phin m tng hp tRNA vi khun do enzyme RNApolymerase xc tc, cn nm men v cc sinh vt a bo do enzyme RNApolymerase III xc tc. S phin m tng hp tRNA sinh vt ni chung thng din ra theo 2 giai on: - Phin m tng hp phn t tin tRNA chung, - Ct b cc intron to phn t tRNA hon thin. Trong trng hp mt gen m ha cho nhiu tRNA th bn phin m u tin c on dn u u 5, on cui u 3 v cc on m nm gia cc tRNA cn c loi b to cc phn t tRNA hon thin. Phn t tin tRNA s c mt endonuclease l ribonuclease P (RNase P) ct ti cc v tr xc nh u 5, to u 5phosphat v mt exonuclease khc l ribonuclease D ct b on khng mang m u 3, to u 3OH. Tuy nhin, u 3 ca tRNA lun c b ba CCA cui cng, l ni axit amin gn vo. Nu ribonuclease D ct sai v tr u 3 th enzyme c hiu CCAnucleotidyltranferase s sa sai u 3 lun c b ba CCA. Mi loi tRNA c hiu cho mt axit amin, do vy, c t nht l 20 loi tRNA. t bo eucaryote, s lng gen m ha cho tRNA nhiu hn t bo vi khun. t bo nm men S. Cerevisiae, ngi ta xc nh c 46 loi tRNA khc nhau, nhng c n khong 360 gen m ha cho cc tRNA ny. Nh vy, tnh trung bnh, mi tRNA c khong 8 gen m ha ring bit. Tuy nhin, con s ny khng th chia u nh vy m thng thng, nhng loi axit amin no c mt trong protein vi s lng ln th s lng gen m ha cho tRNA vn chuyn axit amin s nhiu hn. Cc gen m ha cho mt loi tRNA thng phn b trn cc nhim sc th khc nhau. Do , cc promoter ca cc gen ng vai tr quan trng trong s kim sot qu trnh phin m. 2.3.5- Phin m tng hp rRNA Cc phn t rRNA cng c tng hp t cc gen m ha cho chng trn DNA. Trong t bo procaryote c 3 loi rRNA tham gia vo qu trnh tng hp protein l 16S, 23S v 5S; cn t bo eucaryote, cc rRNA c mt trong ribosome l 18S, 28S v 5,8S.

Qu trnh phin m tng hp rRNA cng xy ra theo hai bc nh tRNA. Phn t tin rRNA c tng hp u tin da trn khun DNA. t bo eucaryote, qu trnh c xc tc nh enzyme RNA-polymerase I. Phn t tin rRNA tri qua giai on ct loi intron to cc phn t rRNA hon thin.
23S
16S Rm Rm
Rm
Rm
1 hoc 2
5S
0 hoc 2
Rm - V tr ct ca enzyme RNase III trn si tin RNA
Hnh 2-14: Nhiu rRNA c mt trong cng mt phn t tin RNA E. Coli
vi khun E. Coli, mt gen thng m ha cho mt s phn t rRNA.
V d 3 loi rRNA l 16S, 23S v 5S c m ha trong mt gen. Khi phin m s to mt phn t tin rRNA chung cho c 3 loi rRNA trn, sau , cc enzyme ribonuclease s thc hin qu trnh ct loi cc intron to 3 phn t rRNA hon thin. Cng c trng hp, trong mt operon m ha cho rRNA c c phn m ha cho c tRNA (Hnh 2-14). 2.4- DCH M (Translation) 2.4.1- c im chung ca qu trnh dch m Dch m l qu trnh tng hp chui polypeptide da trn cc thng tin m ha trn mRNA v trnh t sp xp cc axit amin trn si polypeptide . Qu trnh dch m phc tp hn nhiu so vi qu trnh sao m v phin m. Dch m c thc hin ribosome vi s tham gia ca ba loi RNA. Mi ribosome gm mt n v ln v mt n v nh. t bo vi khun, tiu n v ln cha hai si rRNA l 23S v 5S, cn tiu n v nh cha mt si rRNA 16S. t bo nm men v sinh vt a bo, tiu n v ln cha hai si

rRNA l 28S v 5,8S , cn tiu n v nh cha mt si rRNA l 18S. Khi khng tin hnh tng hp protein, mi n v tn ti tch ri trong t bo cht. Trong thi gian dch m, hai tiu n v ribosome kt hp vi mRNA v tRNA thc hin qu trnh tng hp protein. Hng dch m trn mRNA t u 5 n u 3. Qu trnh dch m c tin hnh khi hi t cc yu t nh s c mt ca mRNA, cc ribosome, tRNA, cc axit amin v cc enzyme cn thit, m bo qu trnh vn chuyn v hnh thnh lin kt peptit. Dch m c thc hin t bo cht. sinh vt procaryote, hai qu trnh dch m v phin m xy ra dng thi. Sau khi si mRNA c phin m c mt on th qu trnh dch m khi u. sinh vt eucaryote, hai qu trnh ny xy ra c lp hai v tr khc nhau. 2.4.2- S hot ha v vn chuyn axit amin Cc phn t tRNA lm nhim v vn chuyn cc axit amin cho qu trnh tng hp protein. Mi axit amin c t nht mt phn t tRNA lm nhim v vn chuyn. Cc axit amin (Aa) c hot ha nh enzyme aminoacyl-tRNA synthetase vi nng lng t phn t ATP. Enzyme ny lm nhim v gn axit amin vo u 3' ca phn t tRNA. B ba cui cng u 3' ca phn t tRNA l CCA. Nhm cacboxyl ca axit amin s to lin kt ng ha tr vi gc ng ribose nucleotide cui. Sn phm trung gian: AaAMP sau khi xut hin lp tc gn vi phn t enzyme cho n khi gp tRNA tng ng th axit amin s c trao cho tRNA hnh thnh t hp Aat-RNA, gii phng AMP. Qu trnh chuyn ha c th biu din nh sau:
Aa Aa AMP
ATP
Aminoacyl-tRNA synthetase
Aa Aa
AMP tRNA
tRNA
ATP

H Amino axit H H N+ H H C R C O O
_
H C
ATP
+ Adenosine
P P P
N+ C H R
O O Oh
C P
O Adenine H
Cytosine P Cytosine O C O P Adenosine Adenosine P
H H
N+ C H R
Amp
+
Aa Amp ni thnh aminoacyl-tARN synthetase
+
Oh Oh H H C P Cytosine P Cytosine P H C P O Adenine H H N+ H H C R C Oh
Aminoacyl tARN synthetase
O O
trna
O Adenine H
Cytosine
Cytosine
Hnh 2-15: S biu din qu trnh gn axit amin vo tRNA ca enzyme aminoacyl-tRNA synthetase

Cc axit amin lun c gn vo tRNA ca n mt cch chnh xc, iu cho thy: trong phn t enzyme phi c nhng vng lm chc nng nhn bit cc c cht ca n mt cch chnh xc. Trong mi t bo phi c t nht 20 loi enzyme aminoaxyl-tRNA synthetase tng ng vi 20 loi axit amin. Qu trnh gn axit amin vo tRNA ca enzyme aminoacyl-tRNA synthetase c biu din Hnh 2-15. 2.4.3- Hng c m v hng ko di si polypeptide S ko di si polypeptide c th biu din nh sau:
O H N H Aa 1 Aa 2 Aa 3 C O tRNA(a) H H N H Aa 1 Aa 2 Aa 3 Aa 4 C O tRNA(b) O tRNA(a) H N H Aa 4 C O tRNA(b) O
Trong qu trnh dch m, ribosome s di chuyn dc theo si mRNA t u 5' n u 3'. Cc b ba m ha cho axit amin c c mt cch lin tc t b ba khi u AUG nm u 5' ca phn t mRNA cho n khi gp cc b ba kt thc u 3'OH. Si polypeptide c tng hp bt u t u cha nhm NH2 n u cha nhm COOH. 2.4.4- Cc giai on ca qu trnh dch m Qu trnh dch m gm 3 giai on: Khi u, ko di v kt thc. 1,- Giai on khi u: u tin, mt methionyl-tRNA synthetase (l mt aminoacyl-tRNA synthetase) gn phn t methionyl vo u 3' ca tRNA
met
to phn t Met-

tRNA . Trong t bo tn ti hai loi tRNA : Loi th nht chu trch nhim mang phn t methionyl n v tr khi u dch m, loi th hai mang methionyl n cc v tr c cha b ba AUG nm gia phn t mRNA. Vi s tr gip ca cc phn t khi ng IF (initiaion factor), tiu n v nh ca ribosome s n gn vo mt v tr chuyn bit ca mRNA, v tr ny nm u 5', khng xa trc b ba khi u AUG, ng thi, phn t Met-tRNA
met met met
cng nh v vo v tr c cha b ba khi u, to nn phc

met
hp gia tiu n v nh ca ribosome-mRNA-Met-tRNA . Ngay sau khi phc hp ny hnh thnh, tiu n v ln ca ribosome s kt hp vo tiu n v nh, to nn h thng dch m: ribosome-mRNA-Met-tRNA 2-16).
met
(Hnh
Cc nhn t khi ng sau tch ra khi h tip tc khi ng cho mt qu trnh dch m khc. Hin nay, ngi ta bit c 3 loi nhn t khi ng procaryote v 6 loi eucaryote. Nng lng s dng khi ng dch m c ly t cc phn t cao nng nh GTP. Phn t mRNA gn vo tiu n v nh ca ribosome khi trong mi trng phi c nng Mg nht nh. 2,- Giai on ko di: Trn ribosome c hai v tr chuyn bit cho tRNA mang axit amin n, l v tr P (peptidyl) v A (aminoacyl). Phn t tRNA mang methionine (Met-tRNA ) u tin n s bm vo v tr P - i din vi b ba AUG, tip sau , mt tRNA th hai mang mt axit amin c b ba m ha tip theo xp vo v tr A. Bc tip theo l hnh thnh lin kt peptit gia nhm cacboxyl ca axit amin gn vi tRNA trn v tr P vi nhm amin ca axit amin gn vi tRNA trn v tr A, ng thi, lin kt gia axit amin vi tRNA trn v tr P mt i, si polypeptide ang hnh thnh lc ny gn vi tRNA trn v tr A. Cng lc , si mRNA dch chuyn mt bc, lm cho tRNA v tr P y ra ngoi, cn tRNA v tr A chuyn sang v tr P. V tr A s tip nhn mt tRNA mang axit amin th ba n. Qu trnh ny li lp li, lin kt peptit th hai c hnh thnh. C nh vy, chui polypeptide c ko di cho n khi xut hin du hiu kt thc, ngha l c b ba kt thc nm i din v tr A.
met +2
30S tiu n v 1F1 1F2 1F3 GTP

1 , 2 , 3 ,
2 3
fMet
1 16S rRNA 5 3 AUG 3

fMet
B i m tRNA
2 5 1 H 30S khi u 3
50 tiu n v v tr P
fMet
1 , 2 , GDP + P v tr A
H 70S khi u
Hnh 2-16: S biu din giai on khi ng dch m procaryote

Met 3' 5' tRNA 5' 5' 5' Val 3' Met 3' Val 3'
C A G
UAC mRNA 5' A U GGU C U A U GAA 3' 5'
UAC CAG A U GGU C U A U GAA 3'
Chuyn dch Met Val Tyr 3' 3' 5' 5' Met Val 3' 5' 5' 5' Tyr 3'
3'
A U G
*
CA GAU G* 5' A U GGU C U A U GAA 3' 5'
A C
CAG
A U GGU C U A U GAA
3'
Chuyn dch
3'
Met Val Tyr 3'
Glu 3' 5'
5'
5'
C U U
A G
CAG * 5' A U GGU C U A U GAA 3'
G* - v tr ny, G c th b methyl ho, n kt cp vi U do tnh linh hot v tr th 3 ny
Hnh 2-17: S biu din giai on ko di chui polypeptide ang tng hp

Cc tRNA mang axit amin (AatRNA) c xp vo ng v tr A trn ribosome nh nhn t ko di EF (elongation factor) vi nng lng t phn t ATP. Lin kt peptit c hnh thnh nh enzyme peptidyl transferase. Mi mt bc dch chuyn, mRNA s tin v pha trc ba nucleotide. 3,- Giai on kt thc: Khi im A trn ribosome tin n i din vi cc b ba v ngha (UAG, UAA, UGA) th s khng c mt tRNA no mang axit amin n na, nn s ko di lin kt peptit s dng li. Kt thc giai on dch m c s tr gip ca cc nhn t kt thc RF (release factor). Nhn t kt thc nhn bit b ba kt thc v lm t lin kt gia cc chui polypeptide mi hnh thnh vi phn t tRNA cui cng, gii phng si polypeptide. Si mRNA cng tch khi ribosome, hai tiu n v ca ribosome tr v trng thi ban u, sn sng cho mt chu k dch m mi. E. Coli, ngi ta tm ra ba nhn t kt thc l RF1, RF2, RF3. RF1 nhn bit cc b ba kt thc l UAG v UAA; RF2 nhn bit cc b ba UGA v UAA. Cc RF ny nhn bit cc b ba kt thc v a enzyme peptidyl transferase ct lin kt gia tRNA vi chui peptit. Cch hot ng ca ring RF3 cha c lm r. 2.4.5- Polyribosome Hin tng nhiu ribosome cng bm vo mt mRNA v thc hin dch m ng thi gi l polysome hay polyribosome. Sau khi ribosome u tin kt hp vi mRNA u 5' thc hin dch m c mt on ngn, th on pha sau, nhiu ribosome khc cng bm vo v cng thc hin dch m ng thi. Mi ribosome m nhn dch m mt on trn mRNA. Ngi ta c tnh khong m nhim dch m ca mi ribosome trn mRNA E. Coli l khong 80 nucleotide. Nh vy, tc tng hp protein tng ln ng k. 2.5- IU HO BIU HIN GEN (iu ho sinh tng hp protein) 2.5.1- Mc ch ca iu ho biu hin gen Mc ch ca s iu ho biu hin gen l nhm iu chnh h enzyme cho ph hp vi cc nhn t dinh dng, tc nhn l ha v mi trng, to s lng v s loi cn thit m bo nhu cu ca t bo l pht trin v sinh sn.

Trong bt k t bo no, tt c cc gen u khng hot ng ng thi, bi v nhu cu protein ca t bo thay i lin tc. Trong c th sng, khng phi loi protein no cng c tng hp vi s lng nh nhau. C loi c tng hp mt cch lin tc trong sut qu trnh sng, v d nh hemoglobin ca mu; c loi ch c tng hp trong mt giai on nht nh no trong chu trnh sng, v d nh hormone sinh trng, hormone sinh sn, ... Nh vy, mt s gen s hot ng nhiu hn, thng xuyn hn, mt s khc ch hot ng nhng giai on nht nh hoc trong nhng iu kin nht nh ca chu trnh sng. p ng y v ng s lng protein cho c th sng, trong c th sng phi c c ch iu ho sinh tng hp protein, lm sao cho lng protein to ra va , khng tha v cng khng thiu. C nh vy, hot ng trao i cht ca c th sng mi m bo bnh thng, ngc li s lm ri lon trao i cht, pht sinh bnh tt hoc t vong. 2.5.2- Cc yu t iu ho biu hin gen thc hin iu ho biu hin gen, u tin, phi c tn hiu gy iu ho, sau , thc hin qu trnh iu ho. t bo procaryote, tn hiu gy iu ho thng l nhng yu t dinh dng hay nhng yu t vt l ca mi trng. S thay i cc yu t dinh dng hay yu t vt l ca mi trng c tc ng lm thay i hot ng ca c th sng, nhm thch nghi vi iu kin mi pht trin. t bo eucaryote, tn hiu iu ho l nhng phn t do t bo chuyn bit pht sinh. V d nh cc hormone c tng hp cc b phn chuyn bit nh tuyn yn, tuyn gip, ... i vo trong mu, em tn hiu n cc m thc hin iu ho biu hin gen. iu ho biu hin gen c th thc hin bng nhiu cch khc nhau nh thay i cu trc ca DNA, tc ng trc tip n cc giai on tng hp protein nh phin m, dch m, ..., qu trnh xy ra rt phc tp. Phn ln sinh vt procaryote u dng n bo, t bo cha c mng nhn, qu trnh phin m v dch m xy ra ng thi trong t bo cht, nn s iu ho biu hin gen c tin hnh ch yu giai on phin m. i vi cc sinh vt n bo, s iu ho l nhanh v nhy, v cc tn hiu iu ho tc ng trc tip ngay t bo. Ngc li, sinh vt eucaryote l nhng c

th a bo: Mi t bo l mt thnh phn ca c th sng, nn phi tun th nghim ngt theo mt chng trnh pht trin chung ca c th, mi b phn c mt chc nng ring bit, s iu ho y mang tnh thng nht cho ton b c th. Thm vo , t bo eucaryote c cu to hon chnh, s phin m xy ra trong nhn t bo, cn s dch m thc hin ngoi t bo cht, do vy, s iu ho biu hin gen phc tp hn nhiu v c tin hnh nhiu giai on. 2.5.3- M hnh iu ho biu hin gen vi khun 2.5.3.1- Cu trc operon B gen ca vi khun c t chc theo operon. Mt operon c mt vng iu khin v vng m ha. Vng iu khin bao gm gen iu ho, promoter v im iu hnh (operator). Vng m ha bao gm mt s gen cu trc nm k nhau, mi gen cu trc m ha cho mt polypeptide. Cc protein c m ha trong mt operon thng c quan h mt thit vi nhau trong mt qu trnh chuyn ha sinh ha no trong t bo. Mt operon c t nht mt promoter, tuy nhin, operon c th c nhiu hn mt promoter v i lc ca cc promoter ny i vi RNA-polymerase l khc nhau. Operator l trnh t DNA, ni m protein c ch (repressor protein) gn vo. Khi nim v operon c F. Jacob v cc cng s ca ng nu ra nm 1961 khi nghin cu s kim sot di truyn hp th ng lactose E. Coli. Vi s khm ph ra c ch iu ho biu hin gen, ng nhn c gii thng Nobel 1965. 2.5.3.2- Operon cm ng - Operon lactose E. Coli Vi khun E. Coli c kh nng s dng ng lactose nh ngun cacbon chnh pht trin. Lactose l mt disacarit, nn khi c mt lactose trong mi trng, vi khun sn sinh ra enzyme -galactosidase thu phn lactose, to thnh cc phn t monosacarit l glucose v galactose thun li cho qu trnh chuyn ha. Ngoi -galactosidase ra, t bo vi khun cn tng hp enzyme permease lm nhim v vn chuyn lactose qua mng t bo. Khi nghin cu s kim sot di truyn hp th ng lactose, F. Jacob pht hin c ch

tng hp cc enzyme hp th ng lactose. Qu trnh th nghim c th m t nh sau: - Khi nui vi khun E. Coli trn mi trng khng c lactose, th ta thy nng enzyme -galactosidase v permease do vi khun sinh ra rt thp: khi a ng lactose vo mi trng nui th nng hai enzyme ny t bo vi khun tng ln rt nhiu. Nhng nu nui vi khun trn mi trng c ng glucose v lactose ng thi th nng hai enzyme nu trn trong t bo thp hn so vi trng hp ch c ng lactose. Khi phn tch tng nng mRNA c mt trong t bo trc v sau khi a lactose vo trong mi trng nui, thy rng: Khi khng c mt lactose trong mi trng th khng xut hin mRNA m ha cho -galactosidase v permease. Khi b sung lactose vo mi trng nui th trong t bo xut hin mRNA m ha cho hai enzyme nu trn. T kt qu th nghim trn, cng vi nhng kt qu thu c v t bin gen, F. Jacob v J. Monod a ra m hnh iu khin operon lactose nh Hnh 2-18. Cu trc ca operon lactose gm: gen iu ho (R), promoter (P), operator (O) v ba gen cu trc l lacZ m ha cho enzyme -galactosidase, lacY m ha cho enzyme permease v lacA m ha cho enzyme transacetylase. Khi khng c mt lactose trong mi trng, gen iu ho thng xuyn tng hp protein c ch. Protein c ch c i lc vi im iu hnh (operator) nn n gn vo im iu hnh, ngn cn khng cho enzyme RNApolymerase thc hin phin m, mRNA khng th tng hp c, operon ng. Khi c mt lactose trong mi trng, nh enzyme permease c sn mng t bo chuyn mt lng rt t vo trong t bo. Khi vo trong t bo, lactose chuyn thnh allolactose (c cha lin kt -1,6). Allolactose l cht cm ng, n lin kt vi protein km hm. Phc hp ny khng c i lc vi operator, nn khng gn ln operator c, lc ny, operon m. RNApolymerase thc hin phin m cc gen cu trc.

R P O Z Y A R: gen iu ho P: promoter O: operon iu ho R P Z Y A
Protein c ch gn vo operator km hm qu trnh phin m Protein c ch (Repressor protein) R P O Z Y A
lac. mRNA -galactosidase
Cht cm ng
Permease
Transacetylase
Hnh 2-18: M hnh hot ng ca operon lactose Phn ln s tng hp cc enzyme hot ng trong cc qu trnh phn gii t bo c s kim sot theo c ch ca operon cm ng. 2.5.3.2- Operon km hm - Operon tryptophan E. Coli Operon tryptophan cng c cu trc tng t nh operon lactose, ngha l, bao gm gen iu ho (R), promoter (P), operator (O) v 5 gen cu trc. Mi gen cu trc m ha cho mt enzyme, xc tc phn ng tng hp tryptophan. Trong t bo, tryptophan c tng hp bng mt chui 5 phn ng, mi phn ng c xc tc bng mt enzyme m ha trong operon. Nm gen cu trc m ha cho 5 enzyme, ln lt c k hiu l trpE, trpD, trpC, trpB v trpA. Gen trpE nm ngay sau vng iu ho, c phin m u tin.

5 gen cu trc Operon tryptophan - gen iu ho - promoter - operator - 5 gen cu trc xc nh 5 enzyme xc tc phn ng tng hp tryptophan
Protein km hm (aporepressor)
mRNA c tng hp (operon m)
Gen cu trc khng c tng hp
Nhn t ng km hm (corepressor)
Hnh 2-19: M hnh hot ng ca operon tryptophan Hot ng ca operon tryptophan khc vi operon lactose ch l protein km hm, bn thn n khng c i lc vi operator nn khi ng ring mt mnh, n khng th gn vo im iu hnh, nn operon m. Ngc li, khi protein km hm kt hp vi cht ng km hm (corepressor), th s c i lc vi operator nn d dng gn vo , lm operon ng. Hot ng ca operon c th m t nh Hnh 2-19. Nh vy, khi lng tryptophan d, operon ng, cn khi thiu tryptophan th operon m. Nhn chung, cch iu ho biu hin gen sinh vt procaryote ch yu c thc hin giai on phin m.

2.5.4- M hnh iu ho biu hin gen eucaryote Cu to t bo eucaryote l hon chnh, c mng nhn ngn cch nhn t bo vi t bo cht. B my di truyn tp trung ch yu trong nhn t bo. Qu trnh phin m c thc hin trong nhn, cn dch m xy ra trong t bo cht. Hai qu trnh ny khng xy ra ng thi nh t bo procaryote. B gen ca sinh vt eucaryote c kch thc ln, cu trc phc tp, phn ln cc gen u cha cc on khng m ha, xen k vi on m ha. Trong nhiu trng hp, phn khng mang m ln hn phn mang m. Do c im cu to nh vy nn vic iu ho biu hin gen rt phc tp. Cc gen iu ho thng c kch thc ln hn nhiu so vi sinh vt procaryote. Cc gen iu ho thng nm cch xa promoter. iu ho biu hin gen c th c thc hin nhiu giai on nh: iu ho bng cch thay i cu trc ca nhim sc cht, iu ho trong giai on phin m, trong giai on dch m v sau dch m. 2.5.4.1- iu ho hot ng biu hin gen bng cch thay i cu trc ca nhim sc cht Nhim sc cht l cu trc lin kt gia protein histon vi DNA. Gen c phin m khi DNA phi gin xon cc b, nu nhim sc cht xon cht li vi cc protein histon th khng xy ra phin m. Kt qu ca nhiu nghin cu cho thy: sinh vt eucaryote, nhiu gen hot ng biu hin mnh, lin tc, thng c sp xp vo mt nhim sc th, nh vy, s thun li cho vic sao chp v phin m. V d cc gen m ho cho hemoglobin (4 gen) c sp xp gn nhau, biu hin ca cc gen ny c iu chnh ph hp vi tng thi k pht trin ca c th. S thay i thnh phn cu to ca cc baz nit trong nhim sc cht cng lm thay i hot ng biu hin gen. V d nh s metyl ho mt s baz nit vng 5' ca gen ng vt s km hm hot ng biu hin gen hay nhim sc th X khng hot ng ngi thuc loi siu metyl ho. 2.5.4.2- iu ho hot ng biu hin gen giai on phin m S phin m c tng cng hoc km hm do s c mt ca mt s nhn t iu ho sau:

1,- S c mt ca trnh t cis trong phn iu hnh ca gen v cc nhn t trans (protein iu ho): on DNA tham gia vo qu trnh iu ho biu hin gen, c cu trc gm hai phn i xng nhau, c gi l trnh t cis. Trnh t ny nm phn iu hnh, l ni tip nhn cc protein iu ho (nhn t trans). c im cu to chung ca nhn t trans l chng bao gm hai vng cu trc: vng chu trch nhim gn nhn t trans vo trnh t cis v vng tc ng ln s phin m. Cc vng cu trc chc nng ny l c lp vi nhau. Ngoi hai vng chnh k trn, nhiu nhn t trans cn c mt s vng ph khc. Nhn t trans gn vo trnh t cis ca DNA nh cc lin kt yu v n tc ng ln s phin m, lm khi s phin m v t tc cao. Nhn t trans c th l mt hormone hay mt protein iu ho. 2,- S c mt ca trnh t khuych i hoc trnh t dp tt: Trnh t khuych i l on DNA c tc dng lm tng s phin m. Khi c mt trnh t khuych i (enhancer) trong gen th s lm tng s biu hin gen. V tr ca trnh t khuych i trong gen l khng c nh, c th nm u 5', u 3' hay ngay trong intron ca gen. Ngoi trnh t khuych i, cn c trnh t dp tt (silence), l on DNA, m khi c mt trong gen th s c tc dng c ch phin m. Nh vy, nu trong gen c cha trnh t khuych i th s phin m s xy ra mnh m, ngc li, nu c trnh t dp tt th s phin m s khng thc hin. 3,- Chn la promoter thch hp: y l kiu iu ho da vo tng tc cis-trans thng gp sinh vt eucaryote. V d in hnh l gen m ho cho -amylase ng vt bc cao, gen ny c hai promoter, mi promoter c hot tnh phin m khc nhau v chu trch nhim phin m cho mt loi mRNA nht nh, c trng cho cc c quan khc nhau trong c th (tuyn nc bt v gan, ty). S la chn promoter hot ng tu thuc vo nhn t trans hin din trong t bo ca cc c quan c kh nng tng hp -amylase. Nhn t trans

c trng s tng tc vi trnh t cis lm cho s tng hp mRNA c thc hin, kt qu l protein c tng hp. 2.5.4.3- iu ho hot ng biu hin gen giai on sau phin m sinh vt eucaryote, si mRNA u tin c tng hp (tin mRNA) phi tri qua giai on hon thin nh to m chp, ct b intron, ni exon, gn ui poly-A hnh thnh si mRNA trng thnh. mRNA i qua mng nhn, vo t bo cht n ribosome, thc hin qu trnh tng hp protein. iu ho hot ng biu hin gen giai on sau phin m ch yu tc ng vo giai on hon thin mRNA. Cc kiu tc ng c th xy ra nh ct cc intron v ni exon khc nhau, gy t bin trn mRNA. Ngoi ra, thi gian tn ti ca mRNA cng c nh hng ng k n s lng protein tng hp. mRNA tn ti cng lu th s lng protein c tng hp cng nhiu. 2.5.4.4- iu ho hot ng biu hin gen giai on dch m v sau dch m giai on dch m, s iu ho biu hin gen thng th hin s bin i cc nhn t khi ng ca qu trnh dch m. iu ho giai on sau dch m thng c th hin s bin i hot tnh ca phn t protein. Sau khi c tng hp, chui polypeptide phi tri qua mt giai on hon thin mi tr thnh phn t protein hot ng. Trong giai on sau dch m, chui polypeptide c th c gn thm cc gc khng c bn cht protein nh ng, phosphat hay mt nhm hu c hot ng hoc c s sp xp, to cu hnh khng gian, ct b hoc gn thm on peptit to phn t protein hot tnh. 2.5.5- S bit ha t bo 1,- Cc t bo bit ho cha thng tin nh nhau: cc sinh vt bc cao, c th trng thnh bao gm nhiu loi t bo khc nhau (da, tht, gan, thn,.. ). S dng k thut lai DNA cho thy, DNA t nhng t bo ca cc m khc nhau ca cng mt c th khng b bin i trong qu trnh bit ho. Ni cch khc, s lng DNA ca t bo bit ho, v cn bn, ging hp t ban u v cha nguyn thng tin di truyn, pht trin thnh c th nguyn vn.

2,- Cc t bo bit ho tng hp cc nhm protein khc nhau: cc t bo bit ho, ngoi vic tng hp cc protein chung cn thit cho cc qu trnh sinh l, chng cn tng hp mt hoc mt s protein ch yu, v d: - T bo c tng hp nhiu myosine, - T bo biu b tng hp nhiu keratine. Nh vy, cng cha thng tin di truyn nh nhau, nhng mi loi t bo bit ho ch s dng mt phn thng tin tng hp ch yu mt s loi protein. Tm li, mc d tt c cc bc trong s biu hin ca gen v cn bn c iu ho, nhng i vi phn ln cc gen, vic khi s phin m l im kim sot quan trng nht.

CHNG III
BIN I VT CHT DI TRUYN
3.1- KHI NIM V BIN D Bin d l nhng biu hin sai khc gia cc c th th h trc vi th h sau hay gia cc c th trong cng mt th h. Gia c th sng v mi trng lun c quan h mt thit vi nhau. Khi iu kin mi trng sng thay i, cc c th sng cng phi c nhng thay i thch nghi vi iu kin mi, pht trin tt hn. Bin d phn nh mi tng quan gia c th v mi trng. Bin d l ngun nguyn liu cho chn lc t nhin v chn lc nhn to. Bin d c th c di truyn hoc khng di truyn cho th h sau. Bin d c di truyn cho cc th h k tip gi l bin d di truyn. Bin d di truyn thng th hin s bin i trong b my di truyn. S thay i c th xy ra trong phm vi mt gen, c th trong mt nhim sc th hoc c b gen. Bin d c th khng di truyn li cho cc th h sau. S thay i ca mt c th hoc mt s c th, nhm thch nghi tc thi vi s thay i bn ngoi, nhng b gen ca chng khng b thay i, nn nhng bin i khng tm thy th h k tip. Nhng c th bin d di truyn cn c gi l nhng t bin. t bin c th l do con ngi gy ra c nh hng hoc do cc tc nhn t nhin. Da vo tc nhn gy bin d, ngi ta chia chng lm hai loi l bin d t nhin v bin d nhn to. 3.2- T BIN 3.2.1- Tc nhn gy t bin B gen lun hot ng trong sut chu trnh sng, n chu s tc ng ca h enzyme trong t bo. Trong qu trnh vn ng nh sao chp, phin

m, mc d c s kim sot rt nghim ngt, nhng nhng s thay i vn lun xy ra. Ngi ta thy rng, trong t nhin, cc gen du c th b t bin, l cc t bin t nhin hay cn gi l t bin ngu nhin. Cc t bin ngu nhin thng xy ra vi mt tn s nht nh v khng xc nh c tc nhn chnh gy t bin. gy mt t bin nhn to, ngi ta thng s dng mt trong cc tc nhn sau: - Dng tia phng x ion ho nh cc tia , , hoc tia X, cc chm tia neutron hoc proton bn vo b my di truyn, gy ion ho cc thnh phn cu to ca DNA, lm bin i DNA. - Dng tia phng x khng ion ho nh tia t ngoi (UV) to cc dimer thymine. Khi chiu tia t ngoi, cc baz thymine nm k nhau trn mt mch s lin kt vi nhau, lm mt lin kt hydro gia cc baz b sung gia hai mch.
CGA TA AC T AG GCT AT T GAT C Uv
AAC TAG CGAT G C TA AT C T =T G
Hnh 3-1: M hnh biu din s hnh thnh dimer thymine - Dng cc cht ho hc gy t bin. Hin nay ngi ta s dng nhiu loi cht ho hc khc nhau lm thay i cu trc ca DNA. C ch tc dng ca mi tc nhn ho hc ln DNA l khc nhau: C loi tc ng vo giai on sao chp, lm sai hng DNA trong giai on sao chp nh bromouracil hay 2-amino purine l cc ng ng ca baz purine v uracil, cng c loi tc ng trc tip ln gen, lm bin i cc baz nit. V d trng hp tc ng ca bromouracil: Do c im cu to ca bromouracil m n c th bt cp b sung vi baz adenine thay v tr ca thymine, ng thi cng c th bt cp b sung vi baz guanine thay v tr ca cytosine (Hnh 3-2), do vy, sau vi ln sao chp t cp AT ban u c th chuyn thnh cp GC.

3.2.2- t bin gen
3
C C N C O C N
H H
C C N C O C N
H H
(a)
Thymine
5-Bromouracil (dng ceton)
H H C H N C C N H N C H O N C N H N H O C C C H H Br H C N C C N H N C N H H O O
H H
O C N C N C
Br C H H
N C
N C
(dng ceton)
A T
(dng enol)
A Bu
Sao chp ln 1
A T
G Bu
Sao chp ln 2
A T
A T
G C
A Bu
Hnh 3-2: 5-Bromouracil tc ng vo giai on sao chp lm chuyn i cp baz AT thnh GC

t bin gen l nhng thay i trong phm vi mt gen, thng th hin bng s bin i mt vi nucleotide hay mt codon, dn n s thay i bn cht hay trnh t sp xp ca cc axit amin trong mt phn t protein no . t bin gen cn gi l t bin im. Cc dng ca t bin gen c th gp l: 1,- t bin im lng hay cn gi l t bin ng ngha: L khi mt nucleotide trong gen b thay i nhng khng a li hu qu. S thay i ny thng xy ra baz th 3 trong codon, nn vn m ho cho axit amin . V d: Codon UCU m ho cho serine, nu b thay i baz th 3 thnh UCC, UCA hay UCG, vn m ho cho serine m khng c g thay i. 2,- t bin sai nghi: Khi t bin, mt codon ny c thay th bng mt codon khc, m ho cho mt axit amin khc, s thay i ny dn n s tng hp mt phn t protein c thnh phn axit amin sai khc, lm thay i bn cht ca protein ban u. V d: Codon AAG m ho cho lysine, khi t bin baz A u codon thnh G, hnh thnh codon mi GAG, m ho cho axit glutamic. Khi tng hp protein ti v tr ca lysine s thay bng axit glutamic.
Cau (His) Gau (Asp) Aau (Asn) Uag (Stop) Uaa (Stop)
UAu
Uac (Tyr)
Ugu
(Cys)
Ucu
Uuu
(Phe) (Ser)
Hnh 3-3: T mt b ba ban u, UAU m ho cho tyrosine c th to thnh 9 b ba khc nhau bng cch thay i mt baz nit

3,- t bin lm thay i khung c: L trng hp m t bin xy ra do mt i hoc chn thm cc baz nit trn DNA lm lch khung c ca cc b ba trong khi dch m. Hu qu dn n s sai khc hon ton v trt t sp xp cng nh bn cht ca cc axit amin trong phn t protein t im xy ra t bin. Cc t bin s cng trm trng nu im t bin cng nm gn u 5' ca mRNA. V d: T mt on gen m ho cho 7 axit amin ban u l: AUG GCC UCU AAC CAU GGC AUA
Met Ala Ser Asn His Gly Ile
Nu mt G v tr th t th khung c s dch chuyn v lm thay i thnh phn v trt t sp xp cc axit amin, tr thnh: AUG CCU CUA ACC AUG GCA UA
Met Pro Leu Thr Met Ala
4,- t bin v nghi: L trng hp, khi mt b ba m ho cho mt axit amin, sau khi thay i, chuyn thnh mt trong ba b ba kt thc l: UAA, UGA v UAG. Kiu t bin ny s lm mt on trong si polypeptide, im t bin cng nm gn u 5' ca mRNA th di on b mt cng ln. 3.2.3- t bin nhim sc th 3.2.3.1- t bin cu trc nhim sc th t bin cu trc nhim sc th l s thay i xy ra trong phm vi mt nhim sc th no . S thay i cu hnh ca nhim sc th thng gp l mt on, o on hoc lp on. 1,- Mt on: L trng hp nhim sc th b thiu mt on no . on b mt c th nm gia nhim sc th, cng c th nm u mt (mt nh). on b mt c th nh, mang mt gen hay mt phn gen. V d: ngi b bnh bch huyt, nhim sc th 22 b mt on.

S mt mt on di nhim sc th thng gy t vong v mt cn bng di truyn b gen. Nguyn nhn gy mt on l do trong qu trnh vn ng, nhim sc th b t ra, sau c ni li nhng khng y nh ban u. cc th d hp t c nhim sc th b mt on c th quan st c di knh hin vi k trc ca gim phn, khi cc nhim sc th tng ng bt cp nhau. Nu c mt on th s thy ngay c nhng on khng tng ng gia hai nhim sc th.
1 2 3 4 5 6 7
on nhim sc th ban u 1 4 5 6 7
(a) Mt on (2,3) 1 2 5 4 3 6 7
(b) o on (3,4,5) 1 2 3 4 5 6 4 5 6 7
(c) Lp on (4,5,6)
Hnh 3-4: S biu din s mt on (a), s o on (b) v s lp on (c) 2,- o on: L trng hp t bin xy ra, khi mt on nhim sc th quay 180. Lc u, on nhim sc th ny b t ra, sau quay 180, ri c ni li. sinh vt eucaryote, on b o c th cha tm ng hoc khng cha tm ng. Nu ch xy ra s o on, th cc gen c th ch b quay ngc m khng mt i. Trong trng hp o on xy ra t bo gii tnh km vi s trao i cho gia hai nhim sc th tng ng trong vng o on, th thng mt na sn phm ca gim phn s mt sc sng (khng th tinh c).

3,- Lp on: L trng hp mt on no ca nhim sc th c tng ln. on lp c th nm cnh nhau hoc xa nhau trn cng nhim sc th. S lp on thng khng gy hu qu nng n nh mt on, nhiu khi s tng on li c li cho s tin ho v to vt liu di truyn mi, lm cho c th c s lng gen di do hn. 3.2.3.2- t bin s lng nhim sc th Hin tng thay i s lng nhim sc th thng gp trong t nhin v thng gp hn c l s tng bi s lng nhim sc th hay cn gi l a bi th. C nhiu kiu a bi th nh a bi th nguyn, a bi th lch. 1,- a bi th nguyn: L s tng nguyn b nhim sc th n bi ln mt s ln nht nh. Nu c th c s nhim sc th l 2n th dng a bi th nguyn s l 3n, 4n, 5n, ... Cc c th a bi th l nh 3n, 5n, ... thng mt kh nng sinh sn hu tnh do cc giao t to nn b mt cn bng v di truyn. cc c th a bi chn nh 4n, 8n, ... th kh nng sinh sn hu tnh ph thuc vo s phn chia gim nhim ca t bo gii tnh. Nu s phn ly cc nhim sc th l cn bng trong giai on gim nhim th s to nn cc giao t c kh nng th tinh, cn ngc li, s to nn cc giao t bt th. Nhiu loi cy n qu v cy cnh l cc th a bi c qu to, hoa to. a bi th pht sinh do tng bi ton b nhim sc th cng ngun gi l a bi th cng ngun. Trng hp a bi th c hnh thnh do tp giao gia cc loi vi nhau, gi l a bi th lai. Trong t bo ca a bi th lai c th c c hai b nhim sc th ca hai loi khc nhau. V d in hnh ca a bi th lai l th nghim ca Karpochenko, khi cho lai cy c ci vi bp ci, nhn c th a bi lai. 2,- a bi th lch: a bi th lch l trng hp t bin do s thay i s lng nhim sc th ca mt cp hoc mt s cp trong b nhim sc th. V d, b nhim sc th l 2n th cc c th t bin a bi th lch c th s c b nhim sc

th l 2n-1, 2n+1, 2n+2, 2n-2, ... Ngha l c th mt i hoc thm vo b nhim sc th mt hoc mt s nhim sc th no . t bin theo kiu ny c th tm thy c thc vt v ng vt. sinh vt lng bi c s lng nhim sc th l 2n. Nu thm vo hoc bt i mt hoc mt s nhim sc th th trong giai on phn chia t bo thng mt cn bng. Trong trng hp t bo gii tnh c s nhim sc th l 2n-1 th trong giai on phn chia gim nhim s to thnh hai giao t c s nhim sc th l n v n-1. Cc t bin a bi th lch thng yu, c th to cc kiu hnh khc nhau v trong nhiu trng hp b cht hoc khng sinh sn. Mt s dn chng v a bi th lch tm thy ngi l: - Hi chng Down: do Langdon Down pht hin ngi c 3 nhim sc th th 21, ngha l, b nhim sc th 2n tr thnh 2n+1 v bng 47 nhim sc th. Ngi bnh c cp mt hi ging mt ngi Mng c, mc chng n n. T l mc bnh tr em do cc b m c tui cao sinh con l nhiu hn. Ngi ta c tnh c khong 1/60 a b b mc hi chng Down do cc b m c tui trn 45 sinh con v khng b nh hng bi tui ca ngi cha. - Hi chng Turner: do Henry Turner pht hin ra. L trng hp n b mt mt nhim sc th gii tnh. B nhim sc th s c 2n-1=45 cp nhim sc th XX tr thnh XO. Nhng ngi b hi chng Turner thng km thng minh, c quan sinh dc km pht trin, c chiu cao thp, khng sinh sn. - Hi chng Klinefelter: do Henry Klinefelter pht hin u tin. L trng hp nhng ngi nam c 3 nhim sc th gii tnh l XXY, nh vy, b nhim sc th s l 2n+1=47. Nhng ngi b hi chng ny khng c kh nng sinh sn, tr tu km pht trin, c b ngc ging n. - Nhng trng hp khc c th gp ngi l: n c 3 nhim sc th gii tnh (XXX) hay nam c 2 nhim sc th Y (XYY). hai trng hp ny, b gen c s lng nhim sc th l 2n+1=47. Nhng ngi c b gen nh vy thng pht trin khng bnh thng, khng c kh nng sinh sn.

3.3- TI T HP Trong qu trnh vn ng, b my di truyn c th thay i. Cc phn t DNA c th ti t hp vi nhau, kt qu l s sp xp cc gen phn t DNA ca th h sau c th khc vi th h trc. iu kin u tin ti t hp xy ra l trn DNA phi c im t. C ch ca ti t hp ni chung cng cha c xc nh r rng. Kiu ti t hp c nghin cu nhiu l ti t hp xy ra gia cc nhim sc th tng ng trong qu trnh phn bo gim nhim. Cc nhim sc th tng ng bt cp hay tip hp vi nhau v c s trao i cho, kt qu to thnh cc giao t c nhim sc th ti t hp (giao t ti t hp). Trao i cho gia hai nhim sc th tng ng c th xy ra mt ln hoc nhiu ln.
Tip hp v trao i cho Cui gim phn I
A A Aa a B bB b a a B b a a B b A B b A b
Cui gim phn II

A B
A A B A a a
B B b b
A A a a
B B
b b
A A C B A a a
C c C c
B B b b
A A a a
C c
B B
C c
b b
Hnh 3-5: M hnh trao i cho gia cc chromatit

Ti t hp c th xy ra u hoc khng u. Ti t hp u l khi s lng cc gen trn hai nhim sc th tng ng sau khi ti t hp l nh nhau, ngha l, s hon i cc on tng ng gia hai nhim sc th tng ng v khng lm mt thng tin di truyn. Ti t hp khng u l trng hp sau khi ti t hp, mt nhim sc th b mt on, cn nhim sc th kia c thm mt on. Ti t hp khng u s dn n kt qu l mt nhim sc th c s gen lp li, cn nhim sc th kia b mt gen. Ti t hp khng u ng vai tr rt quan trng trong qu trnh tin ho ca sinh gii.
PHN II
NHNG NGUYN L C BN CA CNG NGH DI TRUYN

Cng ngh di truyn cn gi l cng ngh DNA ti t hp, v ang tin hnh mt cuc cch mng sinh hc. N ngy cng c nhiu nh hng n y hc lm sng, trong chn nui, trng trt v nhiu lnh vc cng ngh khc. Khi s dng cng ngh di truyn chng ta to ra protein ca ngi vi mt lng ln cn thit cho iu tr bnh. V d nh insuline, hormone sinh trng ngi, cht hot ha plasminogen. Cng bng cch ny ngi ta to ra nhng vaccine phn t ngn nga cc bnh nhim virus nh bnh vim gan B, hay chun on nh trong test pht hin AIDS. V nguyn l c bn thc hin cng ngh di truyn c ngha l phi thc hin k thut DNA ti t hp. tin hnh k thut ny phi tri qua nhiu bc phc tp v tinh vi sau y: - Bc 1: Tch chit DNA t ngun khc nhau, - Bc 2: Chun b phng tin vn chuyn gen, - Bc 3: Chun b cc enzyme ct v gn DNA, thit k vector DNA ti t hp, - Bc 4: Bin np DNA ti t hp vo vt ch, - Bc 5: Theo di biu hin hot ng ca gen ti t hp, - Bc 6: Phn tch cc sn phm ca gen ti t hp. Tri qua hn 30 nm pht trin, khi nim v k thut di truyn c hiu theo ngha rng hn, bao trm hn, n bao gm nhng thao tc khng ch vi tng gen ring l m c nhng phn ln hn ca b gen v nhiu phng php khc khuych i gen (khng qua to dng invivo) nh phng php PCR. Di y chng ti s trnh by cng vic thc hin ca mi bc, tin hnh theo tng chng m u tin l v enzyme.

CHNG IV
CC ENZYME S DNG TRONG CNG NGH DI TRUYN
Sinh hc phn t pht hin v hiu r c ch tc ng ca hng lot enzyme, nh s dng chng nh nhng cng c hu hiu trong vic ct ni v ghp cc gen. Cc enzyme s dng trong cng ngh di truyn c th chia lm bn nhm ln sau y: - Cc enzyme gii hn - Cc nuclease - Cc enzyme kt ni - Cc enzyme tng hp 4.1- CC ENZYME GII HN (RE-Rectriction Enzyme) 4.1.1- Hin tng gii hn v h thng hn ch - ci bin 4.1.1.1- Hin tng gii hn Khi nghin cu v s xm nhim ca thc khun th (phage) vo t bo vi khun, ngi ta nhn thy rng trong mt s trng hp, thc khun th khng th pht trin c trong t bo vi khun v b my di truyn ca chng b ph hu. Ni cch khc, cc vi khun ny khng thc khun th. Th nghim c th m t nh sau: Cho phage xm nhim hai chng vi khun A v vi khun B, cc vi khun ny c nui cy trong hai mi trng thch hp. Sau khi thu hi v phn tch DNA ca phage, ngi ta nhn thy: - chng A: Xut hin nhiu DNA ca phage cn nguyn vn nhng t bo ca vi khun b ph v (do s nhn ln ca phage trong t bo vi khun). - chng B: T bo vi khun khng b ph v nhng DNA phage b ct thnh nhng on c kch thc xc nh. Trong trng hp ny, DNA ca

phage b mt h thng bo v ca vi khun tiu dit khi va mi xm nhp vo vi khun. Hin tng xy ra vi khun B gi l hin tng gii hn. 4.1.1.2- Enzyme gii hn v h thng hn ch - ci bin (Restriction modification system) Vo nm 1962, ln u tin Werner Arber chng minh rng: C nhng enzyme c bit, hot ng trong t bo vi khun, chng c kh nng phn bit DNA ca mnh v DNA l ca phage. Cc enzyme ny hn ch kh nng sinh sn ca phage trong t bo vi khun bng cch phn hy chng mt cch c hiu. Vo nm 1970, Hamilton Smith pht hin vi khun Haemophilus influenzae Rd enzyme gii hn v t tn cho n HindII. Vi nhng th nghim tip theo, Arber v cng s ti trng i hc Geneva pht hin ra nhiu chng vi khun c kh nng phn hu c DNA t loi khc xm nhp vo chng nh kt hp ca hai qu trnh enzyme. Hai qu trnh enzyme c thng nht trong mt h thng gi l h thng hn ch - ci bin, nhm phn hy DNA l v bo v DNA ca mnh. - S hn ch c thc hin nh s hot ng ca cc enzyme ct hn ch (RE - Rectriction Enzyme). chnh l cc endodesoxyribonuclease c bit c th nhn bit mt trnh t nucleotide c hiu trong chui DNA si i v ct c hai si DNA . - S ci bin l s thay i DNA ca bn thn t bo theo con ng c bit ca mi loi, khin DNA khc vi DNA ca loi khc v khng phi l c cht ca enzyme ct hn ch. Nh m DNA ca t bo ch c bo v. S ci bin c thc hin nh cc enzyme metylase (mtyl ho) c trong t bo vi khun. Enzyme ny s metyl ho cytosine v tr (C5) v adenine (trn N ca C6) thuc vng gii hn (chui ch). V d: Enzyme Hind III, sau khi c metyl ho (du chm) adeninee, chui ch ny khng c nhn bit bi enzyme Hind III na, do , DNA khng b ct:

AAGCTT TTCGAA
Vy enzyme hn ch v enzyme ci bin c cng mt i tng nhn bit l chui nucleotide c hiu trong DNA ngoi lai v DNA ca t bo ch, nhm hn ch s sinh sn ca DNA ngoi lai v bo v DNA ca mnh. Tuy nhin, cng c mt s loi enzyme c c hai chc nng hn ch v ci bin. V d: Enzyme EcoRI vi chui ch l: 5----GAATTC-----3 3----CTTAAG-----5 4.1.2- Cch gi tn ca enzyme gii hn Tn gi ca enzyme gii hn thng c 3 hoc 4 ch xut pht t tn ca vi sinh vt m t , enzyme c xc nh v chit tch. - Ch ci u (vit hoa) l ch u ca tn ging ca vi khun t enzyme c trch li. - Ch th 2 v th 3 (vit thng) l ch u tn loi ca vi sinh vt. - i khi c ch th 4 ch chng sinh vt. - Ch s la m ch th t RE c pht hin (c th c nhiu RE c pht hin t mt chng). V d nh EcoRI - c xc nh u tin E. Coli v EcoRV - l enzyme th 5 cng c xc nh E. Coli. EcoRI: 5----GAATTC----3 3----CTTAAG----5 E: Ging Escherichia co: Loi coli R: Chng Ry 13 I: Enzyme c tch ln u tin

Hpa I: 5----GTTAAC----3 3----CAATTG----5 H: Ging Haemophilus pa: Loi parain fluenzae I: Enzyme c tch ln u tin Hind III: 5----AAGCTT----3 3----TTCGAA----5 H: Ging Haemophilus in: Loi influenzae d: Chng Rd III: Enzyme th ba c tch ra BamHI: 5 ----GGATCC----3 3----CCTAGG----5 B: Ging Bacillus am: Loi amyloliquefaciens H: Chng H I: Enzyme th nht c tch ra 4.1.3- Cc loi enzyme gii hn 4.1.3.1- Loi I Nhng enzyme gii hn loi I c trnh t nhn bit c hiu. V tr ct ca cc enzyme loi ny nm ngoi trnh t nhn bit mt khong cch khng nht nh, dao ng t 1.000 n 5.000 nucleotide. Do khng c tnh c hiu trong ct on nn sn phm ca n khng ng nht v khng pht hin c bng in di trn gel. Phn t lng ca chng vo khong 300.000D, cc bn n v khng ging nhau. Cn Mg lm cofactor v ATP cung cp nng lng cho enzyme ny chuyn ng dc theo phn t DNA t im nhn bit n im ct. V d nh EcoK do Meselson v Yuan tch c, ct im cch trnh t nhn bit 1.000 nucleotide hoc xa hn na. 4.1.3.2- Loi II Bao gm nhng enzyme c trnh t nhn bit chui nucleotide c hiu. Khi nhn bit c trnh t , chng ct ngay ti v tr nhn bit.
+2

Phn t lng thp hn loi I v khong t 20.000 n 100.000D. Ch cn Mg lm cofactor v khng cn nng lng ATP. Do c tnh c hiu v ct on DNA nn sn phm thy phn l tp hp nhng on DNA c hiu v cho php pht hin bng in di trn gel agarose. Ngy nay c trn 1.000 RE loi II c tch ra t cc t bo procaryote khc nhau v c hn 70 loi ang c thng mi ha trn th trng. 4.1.3.3- Loi III Bao gm nhng enzyme sau khi nhn bit mt trnh t DNA c hiu th ct v tr cch 20 nucleotide v to ra mt s u ct khc nhau. Tuy rng v tr im ct rt gn trnh t nhn bit ca chng nhng kh on trc c cc im ct . V vy m RE loi III cng nh RE loi I khng c s dng rng ri trong cng ngh di truyn. Trong 3 loi RE ni trn ch c RE loi II c ng dng nhiu hn. 4.1.4- Cc loi RE trong nhm II 4.1.4.1- Tnh c hiu v tr 1,- Trnh t nhn bit ca RE: C th ni cc RE ny l nhng cng c thc th ct cc DNA. S ct ny xy ra mt vng c bit c nhn bit bi enzyme. Mi mt enzyme nhn bit mt on nucleotide khc nhau rt c hiu i vi n v c gi l trnh t nhn bit (chui ch) hay on c ngc xui. c trng quan trng nht ca cc trnh t nhn bit l chng c cu trc i xng nghch o (palindromic), ngha l hai mch ca trnh t hon ton ging nhau khi chng c c theo chiu 5 n 3 mi si n. Cc trnh t nhn bit ca enzyme gii hn c cu to t 4 n 6 i baz. V d nh on DNA c ng khung sau: 5 - C C A G A A T T C A T - 3 3 - G G T C T T A A G T A - 5
+2

2,- Cc kiu ct ca RE: Ct u bng: Mt s RE to vt ct trn phn t DNA ngay chnh gia palindrom, to ra hai on DNA u bng. Sau khi ct, hai u khng c kh nng t kt hp tr li. V d: HpaI 5' G T T A A C C A A T T G 3' 5' 3'
G T T C A A
A A C T T G
Ct u dnh (u lch): Ct bn ny v bn kia ca tm i xng to ra hai u lch nhau mt vi baz. Trong trng hp ny, cc u dnh b sung c th b bt cp tr li. Nh vy, khi cc DNA khc ngun nhng cng c cha trnh t nhn bit c hiu ca mt RE, sau khi b ct s c cc u dnh b sung ging nhau nn cc on DNA khc ngun c th ni li to ra nhng phn t lai. y l c s ca phng php to dng gen, mt phng php thc y s pht trin ca cng ngh di truyn. V d: EcoRI G A A T T C C T T A A G
A A T T G G
C T T A A 3,- S lng on ct:
Mt RE c kh nng thao tc trn ton b chiu di ca mt phn t DNA. S lng on ct ph thuc vo s chui ch ca mt RE c trn

phn t DNA m n thao tc. S dng RE khc nhau, s b ct khc nhau v to ra lng on ct khc nhau. V d: EcoRI GAATTC CTTAAG Nu chui ny c 5 ln trong mt phn t DNA th enzyme EcoRI s ct ti 5 im trn phn t DNA : - Ta s c 5 on nu DNA dng vng - Ta s c 6 on nu DNA dng thng 4,- Kch thc on ct: V vng gii hn c phn b mt cch ngu nhin dc theo mt phn t DNA m c to thnh bng cch t hp 4 loi baz nit (A,T,C,G) nn kch thc mi on ct ph thuc s cp baz ca mi chui ch. Nu vng nhn bit (chui ch) ca enzyme l 6 cp baz s sinh ra cc on c kch thc trung bnh 4 = 4.096 baz 4,1kb. Nu vng nhn bit ca enzyme l 4 cp baz s sinh ra cc mnh c kch thc l 4 = 256 baz 0,26kb. Tuy nhin mt s RE nhn bit cc vng rt him gp trong DNA. V d nh NotI c chui ch (GC\GGCCGC), khi ct cho cc mnh c kch thc khong 1.000kb. PvuI c chui ch (CGAT\CG) cho mnh khong 300kb.
4 6

Bng 4-1: Mt s enzyme hn ch (HC), enzyme ci bin (CB) v chui ch ca n Tn vi sinh vt Bacillus amyloliquefaciens H Bacillus amyloliquefaciens K Bacillus brevisS Haemophilus aegyptium Baccillus subtillis Brevibacterium albidum Brevibacterium luteum Diplococcus pneumoniae Escherichia coli Ry13 Escherichia coli Ry13 Haemophilus influenzae Rb Haemophilus influenzae Rd Haemophilus influenzae Rd -t bin Haemophilus influenzae Rf Haemophilus parainfluenzae Haemophilus parainfluenzae Thermus aquaticusYTI Xanthomonas amaranthicola Xanthomonas badrii Xanthomonas malvaccarum Xanthomonas paravericola Stetomyces achromogenes Stetomyces albus G Nocardia otitids caviarum Providencia stuartii 164 Proteus vulgaris Enzyme (BamHI) BamKI BbrsI (Hae III) Bsu1114 BalI BluI DpaI EcoRI EcoRV Hinb III Hind I (Hind III) Hinf II Hpa I Hpa II Taq I Xam I Xba I Xma I Xpa I Sac II Sal I Not I Pst I Pvu I Chui ch G\GATCC G\GATCC GCTGC GG\CC GGCC TGG\CCA C\TCGAG GATC G\AATTC GAT\ATC A\AGCTT CAC A\AGCTT AAGCTT GTT\AAC C\CGG T\CGA GTCGAC T\CTAGA C\CCGGG C\TCGAG CCGCGG G\TCGAC GC\GGCCGC CTGCA\G HG/CB HC HC CB HC HC HC HC CB HC/CB HC HC CB HC/CB HC HC HC HC HC HC HC HC HC HC HC HC HC
CGAT\CG
Ghi ch: Nhng enzyme c cng chui ch, enzyme no tm thy trc c trong ngoc ( - )

4.1.4.3- iu kin hot ng ca RE a s enzyme bn trn th trng c hot ng t 1 n 100 n v trn l. Mt n v c tnh bng khi lng enzyme ct hon ton 1 g DNA trong 1 gi vi th tch phn ng 50 l, nhit phn ng 37C. Tuy nhin trong thc t, mun ct hon ton lng DNA cn phi tng nng ca enzyme t 5 n 10 ln so vi cch tnh n v ch nh. iu kin phn ng ct ti u l: - C Mg
+2
lm xc tc
- Nhit 37C - pH t 7,2 n 7,8 - Thi gian phn ng t 1 n 2 gi. Cc RE b c ch trong cc iu kin: nng EDTA cao qu hoc s c mt ca phnol (khi tch DNA cha loi ht). 4.1.5- ng dng ca RE trong cng ngh di truyn 4.1.5.1- Phn lp gen Cc RE cung cp mt phng php mi c hiu lc cho vic phn lp gen. V nguyn tc mt b gen DNA phc tp c th b ct thnh nhiu phn on, m t ngi ta c th phn lp mt phn on mang gen c hiu bng cch in di trn gel. Nhng bng in di DNA c hiu c th nghim cho gen nh nghin cu bng cch lai ha vi nhng bn sao phng x ca gen tinh ch t t bo bng nhng bin php thch hp. Gen ton b hoc mt phn ca n c th thu c nhng phn on nh hn bng cch ct on lin tip vi nhng RE khc. T ta c th phn lp c gen mong mun. V d, mun phn lp vng iu ha lac, u tin ct DNA cha gen lac bng Hind III v to nhiu phn on trong c mt on cha lac di 660bp. on ny c tch bng cch in di trn gel. Phn on thu c ct tip bng enzyme Hae III thnh on cha 174bp vn cn cha h thng

iu ha v gen lac. Nu tip tc ct bng nhng RE thch hp ta s phn lp c vng iu ha gen lac. Ngoi ra, cc RE cn c s dng to cc vector chuyn gen. 4.2- CC LOI NUCLEASE 4.2.1- Phn loi Cc nuclease c kh nng phn ct phn t DNA hoc RNA. C ba kiu phn loi cc nuclease sau: 1,- Da vo bn cht ca c cht m n tc dng, ngi ta chia nuclease lm hai loi: - Deoxyribonuclease (DNase), c cht ca n l DNA - Ribonuclease (RNase), c cht ca n l RNA 2,- Da theo kiu lin kt m enzyme thy phn, ngi ta cng chia ra lm hai loi: - Nuclease "a": Thu phn c hiu lin kt phosphodiester gia cacbon (C3) v nhm phosphate. - Nuclease "b": Thu phn c hiu lin kt phosphoester gia cacbon (C5) v nhm phosphate (Hnh 4-1). 3,- Da theo hot tnh enzyme, ngi ta cng chia ra lm hai loi exonuclease v endonuclease: - Exonuclease: Ct cc lin kt phosphoester t hai u ca chui polynucleotide v t ct dn tng nucleotide, chng i hi l phi c nhm OH (3) hoc OH (5) t do hai u chui. 5 3 5 3 3 5 3 5 5 3 5 3 5 3 5 3

- Endonuclease: Thng thu phn lin kt phosphoester v tr bt k bn trong chui v to ra cc oligonucleotide.
OH OH P O 5 CH2 O 1 H O HO P O Nuclease (b) 5 CH2 O 1 H OH 3 O 3 O
Nuclease (a)
UUHnh 4-1: Phn loi nuclease theo lin kt b ct 4.2.2- Mt s nuclease thng dng v ng dng ca n 4.2.2.1- DNase I L mt endonuclease c kh nng phn ct phn t DNA si n hoc si i sau cc baz pyrimidine to hn hp oligonucleotide c nhm phosphat u 5. Kh nng phn ct si n hoc si i trong phn t DNA ph thuc vo s c mt ca Mn
+2 +2
v Mg .
+2
- Khi c mt ca Mn , enzyme ny tn cng vo si i to vt ct u bng hay u dnh. - Khi c mt ca Mg , n tn cng vo mt si trn phn t DNA mt cch c lp. Cc DNase c s dng loi DNA ra khi dch chit RNA nhm lm tinh sch RNA. Ngoi ra, ngi ta cn dng to vt t ngay trn phn
+2

t DNA trong k thut nh du hay pht hin nhng gen ang hot ng trn nhim sc cht, v nng thp chng ch ct nhng v tr siu nhy cm nh nhng gen m, vng va phin m. 4.2.2.2- Nuclease S1 L enzyme c tch t nm si Asp. oryzae, c kh nng phn hu DNA si n, DNA si i. Khng c kh nng phn ct phn t lai DNARNA. Cc enzyme nuclease S1 c s dng nhm cc mc ch sau: - Phn tch cu trc ca cc phn t lai DNA- RNA, - Loi b cc mch n ca u so le to u bng, - Loi b nhng cu trc kp tc trong khi to cDNA. 4.2.2.3- Desoxyribonuclease III (Exonuclease III ) Enzyme ny c tch t vi khun E. Coli. N xc tc phn ng phn ct cc trnh t nucleotide t u 3OH t do ca mt theo hng 3 5. Kt qu l to vng mch n di trn DNA si i, ngoi ra cn c hot tnh 3 phosphate. 3 5 5 3 3 5 5 3
Enzyme ny c ng dng to cc cu trc mch n mt s vng trn phn t DNA sn xut mu d c trng cho tng mch hoc phi hp vi nuclease S1 to t bin mt on ti nhng vng c bit. 4.2.2.4- Enzyme ribonuclease ( RNase A v RNase H) RNase hin in mi ni, enzyme thng mi c trch ly t ty b. c trng ct lin kt phosphodiester ngay sau mt baz pyrimidine nh uracil v cytosine ca RNA mch n. RNase A bn nhit khng mt hot tnh 90C trong mt gi.

c ng dng loi b RNA cn st li trong dch chit DNA hoc loi b cc vng khng bt cp ca RNA trn phn t lai DNA- RNA. RNase H l enzyme xc tc loi b RNA. Sau khi phin m ngc tip tc tng hp mch th hai ca cDNA hnh thnh mch DNA si i. 4.2.2.5- Enzyme phosphodiesterase L mt loi nuclease c trong nc rn, c cht tc dng l DNA v RNA, tn cng t u 3 ca chui polynucleotide.
4.3- ENZYME KT NI
4.3.1- Ligase kt ni hoc hn hai mnh DNA hoc RNA ngi ta phi s dng enzyme ligase trong s c mt ca ATP. Khi to ra mt lin kt ester gia mt mnh cha 5 phosphat v mt mnh cha 3OH. Chng thng c s dng kt hp vi enzyme khc nh kinase v alkaline phosphatase. Thng thng, chp hai mnh u dnh d hn hai mnh c u phng. 4.3.1.1- Ecoli DNA ligase Enzyme ny c trch ly t E. Coli, ch c th xc tc ni hai mnh DNA c u so le. ng dng to DNA ti t hp trong k thut tch dng. 4.3.1.2- T4 DNA ligase Enzyme ny c trch t phage T4. C kh nng xc tc ni hai trnh t DNA u so le v c bit l hai trnh t u bng. Kh nng gn mnh hn 10 ln so vi ligase tch t E. Coli. c a chung trong k thut tch dng. 4.3.1.3- T4 RNA ligase Enzyme ny c trch t phage T4 , xm nhim E. Coli. C kh nng xc tc ni hai trnh t RNA bng lin kt phosphodiester. C cht ca n thng l nhng phn t nh nn c dng nh du phng x u 3 ca cc phn t RNA dng lm mu d phn t.

4.3.2- Cc enzyme dephosphoryl ho ( kh nhm PO4 ) 1,- Alkaline phosphatase: c trch ly t E. Coli hay t rut b. c tnh ca enzyme ny l hot ng pH kim v c kh nng xc tc kh nhm phosphate 5 ca mt chui DNA hoc RNA v cc nucleotide t do.
OH OH 5 3 OH 5 OH 5P 3 OH OH 3 P 5
-3
Mnh ci (DNA) ngoi lai b ct cng RE vi vector Vect b ct bng RE v sau khi kh nhm PO4 bng alkaline phosphatase
-3
OH
5 CH2 O Baz H OH H H
3
OH
OH OH Khe h OH OH 5
5
Khe h
Nucleotide t do kh nhm PO4

3
E kinase chuyn nhm Po4v hn li
UUHnh 4-2: Phn ng ni DNA ngoi lai vi vector kh nhm PO4-3 Alkaline phosphatase c ng dng : - Loi b nhm phosphate 5 trn trnh t DNA hoc RNA trc khi nh du phng x 5 ca chng bng P
32
lm mu d phn t lai.
- Loi b nhm phosphate 5 trn mt vector va mi b ct bi enzyme RE. Nhm trnh vector ny ng kn tr li, khin cho vic ci mnh DNA

ngoi lai vo b cn tr. Trong trng hp ny, si kp c ci vo c mang hai nhm phosphat 5 s to ra hai trong bn lin kt este hnh thnh mt DNA ti t hp kp vng kn c cha hai khe h. S chp ni vector b kh PO4 5 vi DNA ngoi lai to ra DNA ti t hp si kp c hai ch trng (OH) do OH 3 ca on ngoi lai khng to lin kt ester vi OH 5 ca vector kh nhm PO4 (Hnh 4-2) . 4.3.3- Cc enzyme phosphoryl ho - Enzyme kinase (T4 polynucleotide kinase): c chit t trc khun T4 xm nhim E. Coli. C kh nng chuyn nhm phosphate t phn t ATP ln u 5 ca DNA hay RNA b kh phosphate bi enzyme alkaline phosphatase. Enzyme kinase c ng dng nh du phng x u 5 ca DNA lm mu d phn t trong k thut lai hoc chuyn nhm PO4 cc trnh DNA khng c nhm PO4 trong phng php to dng. 4.4- CC ENZYME TNG HP Enzyme sao chp mt axit nucleic: Qu trnh sao chp chui axit nucleic tc l tng hp mt chui DNA hoc RNA c tin hnh theo nguyn tc b sung v i song song. Vic thm mt nucleotide mi c tin hnh theo hng 5 3. 4.4.1- Enzyme sao chp DNA DNA L cc enzyme DNA-polymerase I, enzyme T4 DNA-polymerase, Taq polymerase tng hp chui DNA t mt khun DNA hay cn gi "enzyme ph thuc DNA". 4.4.1.1- Enzyme DNA - polymerase I Enzyme DNA-polymerase c ngui gc c tch t vi khun E. Coli, chng c ba hot tnh: - Hot tnh tng hp DNA theo hng 5 3 v sa cha trong sao chp, - Hot tnh exonuclease theo hng 3 5,
-3 -3 -3 -3
ti

- Hot tnh exonuclease theo hng 5 3. Ngy nay enzyme DNA-polymerase c dng ch yu xc nh trnh t DNA bng phng php didesoxynucleotide ca Sanger, ngoi ra, cn dng tng hp mu d c phng x cao, hoc xy dng cc vector t DNA mch n. Trong thc t ngi ta hay s dng on Klenow l sn phm thy phn ca enzyme DNA-polymerase I, c hot tnh exonuclease theo hng 3 5 v hot tnh tng hp. 4.4.1.2- Enzyme T4 DNA - polymerase C ngun gc t phage T4 xm nhim E. Coli c hot tnh exonuclease theo hng 3 5 . Enzyme ny mnh nn c s dng nhiu tng hp mu d c phng x cao. 4.4.1.3- Enzyme Taq - polymerase Trch li t vi khun Thermocellus aquaticus, l enzyme chu nhit cao, c tc dng khuych i tng hp DNA. Enzyme ny ch yu s dng nhn dng gen trong phn ng PCR. 4.4.2- Enzyme phin m ngc (Reverse transferase) L enzyme c kh nng sao chp b gen RNA ca retrovirus khi k sinh trong t bo ch to ra cDNA, theo chiu 5 3 cn c mt ca mi. y l giai on cn thit to ra ngn hng cDNA. Hin nay trn th trng enzyme phin m ngc c ngun gc t AMV (Avian Myeloblastosis Virus). Enzyme phin m ngc l mt DNApolymerase 5 3, c cc c tnh sau: - L enzyme ph thuc RNA, - Tng hp DNA theo hng 5 3, - C hot tnh RNase.

Chng c ng dng thit lp ngn hng cDNA hoc tin hnh phn ng PCR trn mRNA, ngoi ra, chng cn c s dng xc nh trnh t DNA bng phng php s dng cc didesoxynucleotide ca Sanger. 4.4.3- Cc enzyme tng hp RNA (RNA-polymerase) C ba loi RNA polymerase c s dng nhiu nht hin nay l SP6 RNA-polymerse c ngun gc t phage xm nhim Salmonella typhimurium. T3, T7 RNA-polymerase c trch ly t phage T3 v T7 xm nhim E. Coli. Cc enzyme ny hot ng trn si khun DNA xc tc s tng hp RNA theo hng 5 3. Qu trnh tng hp khng cn mi nhng khun DNA phi mang promoter c trng ca phage. Cc enzyme ny c ng dng : - tng hp cc mu d RNA nh du phng x trong phng th nghim. - Nghin cu bn phin m RNA ca mt DNA c dng ha nh phng php PCR. - Xc nh trnh t DNA c gn trong mt vector c mang cc promoter c trng ca phare SP6, T3 , T7. Ngoi ra ngi ta cn dng enzyme ny tng hp lng ln RNA t DNA c ni ngay sau promoter thch hp. 4.4.4- Enzyme terminal - transferase Enzyme ny c trch ly t tuyn c b. C cc c tnh: - Terminal-transferase xc tc gn cng mt loi nucleotide vo u 3OH t do ca phn t DNA to ui polynucleotide. Chng c ng dng : - Thm ui polynucleotide to u sole cho phn t DNA dng trong k thut to dng. - nh du u 3 (OH) ca phn t DNA xc nh trnh t axit nucleic theo Manxam v Gilbert.

CHNG V
VECTOR CHUYN GEN
5.1- KHI NIM V VECTOR 5.1.1- S chuyn gen Vic nghin cu mt gen xc nh sinh vt eucaryote gp phi nhiu kh khn, v gen ch l trnh t nh nm lt trong ton b gen c kch thc ln. thu nhn lng gen tinh sch vi hm lng ln ngi ta phi chuyn v to dng gen . chuyn gen t t bo A (t bo cho) sang t bo B (t bo nhn) ch c th thc hin bng cch nh cc gen cn chuyn vo mt yu t trung gian c gi l on dn. Nhng on dn ny c kh nng hng dn, vn chuyn cc on gen cn nghin cu vo t bo nhn. on dn ny chnh l cc vector chuyn gen. 5.1.2- Vector Vector l phn t DNA nh (ngn) dng thng hoc dng vng, trong , ngi ta s ci mt mnh DNA (gen qu) cn nghin cu. Nhng mnh DNA c ci gi l on ci (insert) hoc DNA ngoi lai hoc DNA l. Cc phn t DNA nh ny thng l nhng thc khun th hoc cc plasmid m trong b gen ca chng c tn hiu cn thit cho chng ti bn, nhng chng li khng bit ti bn (sinh si ny n) mt mnh. Chng cn a vo trong cc t bo ch (v d nh t bo vi khun chng hn). 5.2- NHNG YU CU TI THIU CA MT VECTOR CHUYN GEN Vector phi c mt vng nhn bit i vi mt enzyme gii hn loi II. Vng ny s l vng ci lp mt DNA (gen cn nghin cu) c x l bi cng enzyme gii hn. Tht vy, mi mt RE to ra mt u so le ring, c kh nng gn vi mt on DNA mang u so le tng ng. Hn na, cc vng nhn bit ny thng c t vo gia cc gen ch th tin theo di

s biu hin hot ng ca gen ti t hp. Thng thng, l nhng gen khng khng sinh, hoc gen m ha tng hp enzyme c kh nng chuyn ho c cht to mu d pht hin trn mt thch. Vector phi tn ti trong t bo ch qua nhiu th h v t gy xo trn trong t bo ch. Vector c kch thc cng nh cng tt thu nhn lng DNA ti a v d bin np vo t bo ch. Vector phi c kh nng t sao chp tch cc trong t bo ch, khng ph thuc vo s sao chp b gen ca t bo ch. m bo s di truyn bn vng ca mt DNA ti t hp. Chng khng c kh nng sng st ngoi t bo ch v khng chuyn vo t bo ch khc bng con ng tip hp. C trnh t iu ho to iu kin thun li cho vic phin m ca gen c a vo. Kh nng bin np ln ngha l c kh nng thm tt vo t bo ch ca vector mang gen ti t hp. D theo di s biu hin ca gen ti t hp, yu cu ny l cn thit cho vic pht hin dng cn tm. Tinh ch d dng vi khi lng ln bn sao ca gen gn vo vector. Ngy nay cc vector chuyn gen ngy cng hon thin v tin li cho vic s dng. Khng c vector no ton nng cho s chuyn gen m cn c s la chn ty i tng v ty kch thc on gen cn to dng. Chng c cu to vi nhiu tnh cht chuyn bit mang c cc trnh t nucleotide nh mong mun. 5.3- MT S NG DNG CA VECTOR CHUYN GEN - Mt ng dng u tin l tin hnh mt qu trnh c gi l s tch dng nhm khuch i lng ln bn sao DNA xc nh. - Nghin cu s biu hin ca mt on DNA cha bit.

- a gen m con ngi cn nghin cu vo t bo ch. - Sn xut protein t gen c to dng. - Sn xut RNA vi khi lng ln t DNA to dng. 5.4- CC VT CH THU NHN CC VECTOR CHUYN GEN Mi thao tc vi DNA v vic to dng cc phn t DNA ti t hp c thc hin trong ng nghim. Nhng vic to dng mt gen momg mun phi c thc hin trong t bo sng. Do vy, vic bin np, tip nhn v th hin gen trong t bo sng l cc k quan trng trong qu trnh nghin cu. T bo nhn gen ti t hp phi l mt c th ph hp vi gen v phi c nhng bin php chn lc cc t bo ch tip nhn gen. Ngy nay, ngi ta bit c bn loi vt ch thu nhn cc vector chuyn gen l: - Vt ch l vi khun E. Coli v cc vi khun khc, - Vt ch l nm men v nm mc, - Vt ch l t bo thc vt bc cao, - Vt ch l t bo ng vt c v. 5.5- CC LOI VECTOR CHUYN GEN
DNA ti t hp
mRNA
S biu hin S tch dng
UUHnh 5-1: Nhng ng dng DNA ti t hp
Da vo ngun gc ngi ta phn ra: - Cc vector l nhng plasmid, - Cc vector l phage, - Cc vector lai nhn to nh: cosmid, bluescript. Da vo chc nng ngi ta phn ra: - Vector tch dng (c km theo s biu hin hoc khng biu hin ca protein - Hnh 5-1), - Vector biu hin (vector tng hp). 5.5.1- VECTOR CHUYN GEN L PLASMID Cc plasmid l nhng mu DNA nh, ngn, dng vng (khp kn), si i nm ngoi nhim sc th, c tm thy u tin trong t bo mt s vi khun. Chng sao chp c l nh mt s enzyme c mt trong t bo vi khun v khng ph thuc vo s sao chp nhim sc th vi khun. Ty cc kiu ca plasmid m s bn sao plasmid bi vi khun s khc nhau. Mt s plasmid ch c mt bn sao duy nht v chng t ti bn ch mt ln trong mi ln phn bo. Mt s khc c s bn sao ln v chng ti bn c nhiu ln trong mi chu k phn bo. Nhng plasmid c t 10 n 100 bn sao trong t bo ch c xem nh plasmid c bn sao cao. Plasmid khc c t 1 n 4 bn sao trong t bo ch, c xp vo nhm c bn sao thp. Trong sinh vt eucaryote, plasmid ch c trong t bo nm men. Mi plasmid u c mt chui m di truyn (sequence) mang chc mng t ti bn DNA. Nu khng c v tr khi u phin m (ori) ny, DNA khng th t ti lp trong t bo ch. Vi tnh cht t ti bn, plasmid l mt vector chuyn gen nhn dng DNA cn thit. Do kch thc nh nn plasmid ch cha rt t gen chn lc, thng c tnh chn lc l khng khng sinh. T khi c pht hin n nay, cc plasmid khng ngng c ci tin v ngy cng c c thm nhiu c tnh qu cho vic to dng.

Trong phng th nghim, ngi ta s dng cc plasmid nhn to m c to ra t cc plasmid t nhin v ci thm mt s chui DNA. 5.5.1.1- Cc plasmid th h th nht Th h u tin l cc plasmid tm thy trong t nhin pSC101 (Stalay-Cohen), ColE1 gp phn u tin vo lch s to dng. Tuy vy cc plasmid ny c rt t nhng c tnh cn thit. Sau ny, cc nh nghin cu tm ra cc plasmid nhn to th h hai, ba bng cch tp trung nhiu c tnh qu ca nhiu plasmid t nhin vo mt cu trc duy nht. 5.5.1.2- Plasmid th h th hai pBR322 - l plasmid c s dng rt ph bin vo nhng nm 1980 nhn dng trong t bo E. Coli. N c tm ra vo nm 1977 bi Bolivar Rodrigues. Cc plasmid th h th 2 c cu to phc tp hn bt ngun t plasmid nh v c cu to thm nhiu on gen qu (Hnh 5-2).
R
4 im nhn bit Tet

R
Amp
3 im nhn bit 6 im nhn bit
pBR322
Ori
6 im nhn bit
Hnh 5-2: Cu to plasmid pBR322 pBR322 c kch thc 4.363bp v hai gen khng thuc, mt chng chu c ampicilline (ampR) v mt chng chu c tetracycline (tetR) C nhiu im nhn bit bi enzyme ct hn ch v trong s c nhiu im nhn bit nm trong gen khng khng sinh.

V d, gen khng ampicilline c ba trnh t nhn bit bi ba enzyme ct hn ch l PstI, PvuI, ScaI. Cn gen khng tetracycline c su im nhn bit l: EcoRV, BamHI, SphI, SlaI, XmaIII, NnuI. Vic ci DNA l c tin hnh mt trong hai gen khng thuc. Nu ch cn mt gen khng vi khng sinh l do mt trong hai gen khng nhn on ci. iu ny cho php chn la cc plasmid ti t hp. Mt ng dng na ca pBR322 l chng c s bn sao ln. Thc nghim cho thy c 15 plasmid ti t hp c bin np trong E. Coli, s lng ny c th tng ln t 1.000 n 3.000 bn sao trong iu kin nui cy tt. 5.5.1.3- Cc plasmid th h ba y l cc plasmid mnh v a nng, tin s dng cho nhiu loi RE khc nhau vi hng chc trnh t nhn bit ca chng c ni tip nhau thnh mt on di gi l polylinker. Kch thc nh, sao chp nhanh trong t bo vi khun, to s lng bn sao ln. Cc plasmid th h 3 c chia lm 3 nhm ln: - Dy pUC nh: pUC18 , pUC19 (Hnh 5-3) - Dy Gemini: Plasmid pGEM3 , Plasmid pCR 2.1 - Nhm cc plasmid Bluescript 1,- Dy pUC: pUC l plasmid ca University California, c kch thc 2,6kb v c mt s c trng sau: - C mt mnh operon lacZ, cc vi khun ch yu s dng vi pUC dng tng hp enzyme -galactosidase. - C vng polylinker vi 13 vng gii hn ca 13 enzyme ct hn ch. - pUC18 , vng hn ch (EcoRI, SacI, KnpI, XmaI, BamHI, XbaI, SalI, HindI, AccI, BspmI, PstI, SphI, Hind III).

- pUC19 , vng polylinker c trnh t ngc li (Hind III .....EcoRI). - C mt gen bn vi ampicilline, gen ny m ho cho protein (enzyme) vn c tit ra trong khong ngoi bin mng sinh cht ca vi khun.
Polylinker
Gen khng ampicilline
pUC18 pUC19
Operonlac
Hnh 5-3: Cu to plasmid pUC S hin din ca lacZ to thun li cho vector ti t hp bng cch quan st cc vi khun trn thch chn vi khun c kh nng tip nhn mt plasmid. S c mt ca gen khng ampicilline cho php pUC c tin hnh nui cy trn mi trng c ampicilline (Hnh 5-3). u im: Kch thc nh, d bin np vo t bo vi khun. Vng polylinker cho php gn xen bt k trnh t DNA l no, hoc c th cho php to dng on DNA c hai u dnh khc nhau m khng cn cht gn. 2,- Plasmid pGEM3 : (Hnh 5-4)
6
RN A RN
Gen Ap
Hind III
pGEM3 EcoRI Promotor T
Hnh 5-4: Cu to plasmid pGEM3 (Gemini) Kch thc khong 3kb, mang gen khng ampicilline (AmpR) v vng polylineker gm 13 trnh t nhn bit bi 13 enzymee ct hn ch tng t nh vng polylineker ca pUC19 .

Hai promotor c trng cho RNA-polymerase Sp6 v T7 hai bn vng polylinker. V vy c u im l cho php phin m on DNA gn trong vector thnh nhiu RNA, m cc RNA ny thng c dng lm mu d, hoc dng trong nghin cu cu trc chc nng ca RNA. 3,- Nhm cc plasmid bluescript: Bluescrip l vector lai nhn to phage si v plasmid v vy n c kt hp tt c nhng u im ca cc phage v u im ca cc plasmid, c th xem l nhm c tim nng nht hin nay. Cu to: Bluescript c cu to dng vng khp kn, kch thc vo khong 2.961bp (Hnh 5-5), bao gm:
f 1 (-)
Ssp I 19 Ssp I 2850 Xmn I 2645 Sca I 2526

pi c i
lli
n
k h
i u
Ssp I 442
f 1 (+
)k
i u
Nae I 330
Am
Pvu I 2416
BssH II 619 Kpn I 657
lacZ
T7
Vng polylinker SK
pBluescript II SK (+/-) 2961 bp
Sac I 759 BssH II 792
T3
Pvu II 977
Co l E1
kh
i u
Afl III 1153
Hnh 5-5: Cu to pBluescript SK (+/-) - Mt mnh lacZ u 5 ca gen ny c ci sn polylinker gm 21 vng gii hn: KpnI, ApaI, DraII, XhoI, AccI, HindII, SalI, ClaI, HindIII, EcoRV,

EcoRI, PstI, SmaI, BamHI, SpeI, XbaI, NotI, XmaI, SacII, BstXI, SacI hoc ngc li. vng polylinker ngi ta cn tm thy mt vng nhn bit tng i him (NotI) m khng c dy pUC. Vi 2 promoter T3 v T7 thu c on ci RNA cng chiu hoc ngc chiu qua vic s dng RNApolymerase ca phage T3 nhn bit promoter T3 hoc ngc li. - Mt mnh DNA ca phage ty mc ch s dng c th l phage M13 hoc phage f1 .Vng ny s s dng khi ngi ta mun DNA ti bn di dng si n hoc si i, ngoi ra vng ny cn cha im khi u cho s ti bn (ori). Song s ti bn ch c th xy ra bi s ng gy nhim vi mt virus tr gip (virus helper). Virus ny s b khuyt nhng chc nng thiu bng cch mang n nhng gen m ha cc enzyme cn thit cho s ti bn. Ph thuc vo s ci t ca mnh DNA ny so vi chiu ca gen lacZ, ngi ta s c vector bluescipt + hoc . Cp ny cho php nhn c si cng chiu hoc ngc chiu. Mt gen chng chu c ampicilline, gen ny cho php chn la tt c nhng vi khun st nhp mt bluescript. Trong vector bluescript cn c im khi u sao chp colEI ori, chui ny cho php nhn bn ca phagemid (bluenscript) trong E. Coli nh plasmid. Ging gc E. Coli c s dng thng l XL1Blu, trong b my di truyn ca chng c cha gen m ha tng hp tetracycline. iu ny cho php d dng chn la nhng vector st nhp vo vi khun. ng dng: Ty theo iu kin thao tc ngi ta c th s dng bluescript nhn bn mt on DNA si n hoc si kp hoc sao chp RNA invitro sn xut u d lai ha hoc to ngn hng cDNA. 5.5.2- CC VECTOR CHUYN GEN L PHAGE Phage (thc khun th) l virus xm nhim vi khun lm phn gii vi khun. Vic s dng phage lm vector chuyn gen c nhiu u im hn so vi vector l plasmid: - D xm nhp vo vi khun, - Kh nng nhn ln nhanh trong t bo ch,

- Kh nng tip nhn on DNA l ln hn plasmid. Tuy nhin, vic s dng phage c nhiu bt li nh: - Thao tc ghp DNA l phc tp, - DNA ti t hp khng to thnh khun lc nh DNA ti t hp l plasmid, m thnh a phn gii xut hin trn mt thch ph y vi khun Phn ln cc nhm phage s dng lm vector u bt ngun t phage thuc th h th nht. 5.5.2.1- Phage (th h u) Cu to (Hnh 5-6) gm hai phn: Phn u cha DNA v c ng gi trong v protein. Phn ui cho php virus c th t c nh trn cc t bo ch l vi khun: - DNA ca phage: Si i, mch thng, di 48,5kb gm hng chc gen. u tn cng cu DNA l si n gm 12 nucleotide v chng c gi l nhng u dnh. - Cng nh tt c virus, DNA ny c bao bc trong v protein. Phage sinh si ny n theo hai cch: - Chu k tan: phage c sinh si ny n trong vi khun. Cc phage mi c to ra s i ra khi vi khun bng cch lm tan vi khun ny.
DNA
Hnh 5-6: Cu to ca phage
ui

- Sinh tan: L mt kiu sinh sn khc ca virus, phage khng lm tiu tan vi khun, thay v t sinh sn trong t bo cht, phage st nhp DNA ca mnh vi DNA ca vi khun. 5.5.2.2- Cc bin hnh ca phage (phage th h sau) Cc phage thuc th h th hai rt a dng, mi mt loi thch ng vi mt mc ch s dng. 1,- Gim mt s vng hn ch ging nhau: Phage t nhin (cha bin hnh) gi l phage hoang di c cha nhiu vng hn ch ging nhau nh: - 5 vng EcoRI - Nhiu vng Hind III Khng th ct vector ny bng EcoRI hoc HindIII ci vo y mt mnh DNA l. Ngi ta bin hnh loi phage ny ch cn cha mt vng nhn bit ca EcoRI to ra vector ci v hai vng EcoRI to ra vector thay th. V d: - NM607 l vector ci cha on ci DNA di 9kb v tr ct bi EcoRI trong gen CI. - charon 16: DNA c ci vo v tr EcoRI lm c ch gen lacZ. - EMBL4 l mt vector thay th, on DNA thay th di 23kb. 2,- Lm khuyt nhng phn khng cn thit: (Hnh 5-7) Phage ch c th nhn c DNA l trong mt chng mc rt hn ch. Kh nng ca n s b gim rt nhiu khi di genon (b gen) ln hn 105% (51 52kb) hoc nh hn 78% (38 38,5kb) so vi di ban u. Ngi ta tm cch tng chiu di ca on c ci bng cch gim i nhiu hoc t chiu di nhng phn khng cn thit ca genon ca phage . Trong s cc vector b 1/3 phn trung tm gia gen J v N. Trong khi gi li u 5 (tay tri) vn m ho cho protein u, ui. Cng nh u 3 (tay phi) vn m ho cho protein thit yu cho s ti bn v chu k tan.

Cc gen m ho protein u ui Cc gen m ho protein ti bn, chu k tan
DNA l
5 17kb Tay tri 35kb Tay phi
0kb
48,5kb
Phn trung tm
Hnh 5-7: Phage bin hnh Phn trung tm gia gen J v N khng cn thit cho s sinh sn ca phage c th c thay bng DNA l. Cc u mnh 5 v 3 u l u dnh. 3,- Ci mt mnh ca operon lacZ: Mc ch l chn ra phage ti t hp m em li kh nng phn bit bng mt cc phage c st nhp mt on ci vo trong b gen ca chng.
Br
5
O Cl Br
4 5 3
NH Ch2 OH O HO OH OH X-Gal (khng mu)
Enzyme -galactosidase
Ch2 OH O OH HO OH
NH X(5 Brom-4 Clo3 Hydroxyindole) mu xanh
OH Galactose
Hnh 5-8: C ch phn ng thy phn X-Gal bi enzyme -galactosidase Bt u t phage ngi ta cu to nn (bin hnh) phage gt11 l mt vector biu hin. Phage ny c ci vo gen lacZ mt DNA cn to

dng. Cc phage gt11 to ra c cc vng tan trng nu chng c ci DNA l vo gen lacZ (gen chu trch nhim tng hp -galactosidase). Cc phage gt11 to ra vng tan xanh nu nh chng khng tip nhn on ci DNA l. pht hin cc phage ti t hp, ngi ta cho vo mi trng cha phage ti t hp (mi trng nui cy) mt ng X-Gal (5brome-4chloroindolyl--galactopyroside). Ging nh ng lactose, n b thu phn bi enzyme -galactosidase. Ngi ta c th d dng pht hin khi c mt ca enzyme -galactosidase (Hnh 5-8). 5.5.2.3- Cc vector chuyn gen M13 c tnh cu to: L phage th si, xm nhim c trng E. Coli. DNA ca phage mch n, c kch thc 6,4kb, trong c khong 10 gen. ng dng: Do u im ca loi vector ny l to c mt lng ln phn t DNA ch mang trnh t ca mt mch, nn chng thng c dng xc nh trnh t nucleotide trong phng php Sanger. 5.5.2.4- Cc bin hnh ca phage M13 - Vector M13 mp2 l dng n gin nht, c mt hoc hai trnh t nhn bit bi EcoRI nhn on ci c u dnh c ct bi enzyme EcoRI. - Vector M13 mp7 c to thnh bng cch a thm v tr ct hn ch vo gen lacZ ca vector M13 mp2. Vector ny c 4 im nhn bit bi bn enzyme ct hn ch: EcoRI, BamHI, SalI v PstI. 5.5.3- CC VECTOR CHUYN GEN KHC 5.5.3.1- Cc cosmid 1,- c tnh cu to: (Hnh 5-9) Cc cosmid l nhng vector lai nhn to t mt plasmid vi cc trnh t cos ca phage (c s dng t nm 1978). Cc trnh t cos ny iu khin s ng gi DNA ti t hp vo u ca phage. Khi bao gi cc vng cos u b ct, ch cn mt phn DNA ca cosmid c gii hn bi cc u dnh vi

on ci DNA l c bao gi. Trong phn ng bao gi in vitro, cc protein cn thit cho s to thnh u v ui phi c thm vo cho cc phage c th t hp thnh. Cosmid l nhng plasmid c cc vng gii hn m ti y, ngi ta c th ci lp DNA l v mt gen chng chu ampicilline. Kch thc cosmid 5kb do , n c th nhn c on ci 3545kb v c th ti 47kb (nh chng ta thy Mc 5.5.2.2, u ca phage c th bao gi c 52kb). Vo thi im gy nhim E. Coli, DNA ti t hp c phng vo vi khun. Trong vi khun cc u dnh s bt cp to ra cosmid ti t hp khp kn dng vng v ti bn nh mt plasmid. 2,- u im v ng dng ca cosmid: Cng nh phage, cosmid cho kh nng xm nhim t bo vi khun ln, nhn on ci c kch thc ln.
Vng ct bng RE ci DnA ngoi lai
5 kb
Cosmid
ApR
Cos
Hnh 5-9: Cu to cosmid Trong t bo ch, n t nhn bn nh plasmid. Cho nn ngi ta nhn c nhng khun lc, ch khng phi a phn gii trn mt thch, thun li cho vic quan st. Vi nhng tin li trn, ngi ta dng cosmid tch dng t nhng gen ln to ngn hng genom (b gen). Nhng vi khun mang vector ny c kh nng chng chu vi mi trng c ampiciline. Tuy vy vic ng dng cosmid tch dng cng rt kh khn v vt v.

5.5.3.2- Cc nhim sc th nhn to ca nm men YAC (Yeart - Artificial - Chromosomes) Do nhu cu to dng vi nhng trnh t DNA ngy cng ln, cc nh nghin cu tm ti pht trin nhng vector ngy cng mi. Ngy nay ngi ta to ra YAC cho php to dng vi nhng on DNA di 1501.000kb, trung bnh l 350kb. Bng nghin cu cho thy nm men (Saccharomyces Cerevisiae), nhim sc th mun nhn i v phn ly tt cn ba trnh t: - 2 TEL (telomere): Trnh t u cui ca NST - CEN (centrmere - tm ng): Trnh t trung tm ca NST, m bo s chia i v i v 2 cc ca t bo - ARS (autonomously replicating sequence): Trnh t sao chp t ch tng t nh ori plasmid Da vo ngi ta cu to NST nhn to c ba trnh t ni trn vi cu to gm 2 cnh tay, gia 2 cnh tay ngi ta c th ci t mt on DNA cn to dng vi kch thc khong 150 n 1.000kb. Trn mi cnh tay gm cc gen nh du di truyn chn lc cc t bo nm men c cha YAC v cc chui tn cng c chc nng telomer on cui ca NST. Mt trong hai cnh tay mang mt mnh DNA hot ng nh mt tm ng v mt ngun ti bn ori. Vic ci DNA l vo gen m ha cht c ch tRNA vn chuyn tyrsine s lm bin i mu sc khun lc t bo gc c mang gen h phch (khun lc t trng sang ) khi c mt ca DNA l. y l du hiu v s hin din ca YAC ti t hp trong t bo nm men. Cc nhim sc th ny c a vo t bo ch (nm men), n c nhn ln nh NST khc trong t bo nm men v ta c c nhng gen mong mun. 5.5.3.3- Cc nhim sc th nhn to ca ng vt c v 1,- Cu to: Tng t nh YAC v bao gm: - Trnh t TEL - Trnh t CEN - Trnh t ARS

Nhng khc vi YAC l trnh t TEL v CEN c ngun gc t ngi. iu ny cho php MAC (Mammalian Artificiel Chromosomes) vo t bo ng vt c v v gi c n nh trong t bo. 2,- Mc ch s dng: Ch yu s dng nghin cu cho cc t bo bc cao m chng ta khng s dng c vi t bo nm men. Khi a NST vo t bo ch, n cng tn ti c v nhn ln trong t bo ch. 5.5.4- Cc vector l virus ca eucaryote L nhng vector t virus v t bo tip nhn vector ny l t bo eucaryote. N c cu to ging nh phage . Tuy nhin cc loi vector ny c tnh c hiu cao v ngi ta xy dng da trn nhng hiu bit c th.

CHNG VI
CNG NGH DNA TI T HP V S TCH DNG
6.1- CNG NGH DNA TI T HP 6.1.1- DNA ti t hp 6.1.1.1- Khi nim 1,- DNA: DNA l ch vit tt ca axit 2-deoxyribonucleic, c nh sinh hc ngi Thy in Miescher tm ra vo nm 1869 trong nhn t bo bch cu (t bo m). u tin ng gi n l nuclein c ngha l hch nhn. Sau , ng pht hin ra n c bn cht axit v gi n l axit nucleic. Sau hn 80 nm cng vi s tranh ci ca nhiu nh khoa hc, n nm 1952, ngi ta mi cng nhn DNA l vt cht di truyn. DNA gm 3 thnh phn chnh: baz nit dng purine v pyrimidine (A,T,C,G), ng pentose (ng 5 carbon) v axit phosphoric (H3PO4). Ba thnh phn ny c t l 1:1:1 v chng lin kt vi nhau to thnh mt nucleotide:
Oh Ho P ch2 5 H H 3 Oh O O H A(t,c,g) H
Hnh 6-1: Cu to mt nucleotide Cu trc ca DNA c Watson-Crick khm ph ra. l chui xon kp cong nh nhng, c cu trc khng gian 3 chiu, gm hai si song song,

i xng v b sung cho nhau theo mt qui lut nghim ngt: A bt cp vi T v C bt cp vi G nh cc lin kt hydro. Cu trc xon kp ca WatsonCrick l chic cha kha m ra nhng k thut ca s sng. 2,- DNA ti t hp: Vi cu trc c bit ca phn t DNA v s bt cp nghim ngt ca cc baz nit, nhiu nh nghin cu ny ra tng: nu to ra c nhng ui mt trong hai si ca phn t DNA khc nhau th chng c th ni li vi nhau nh bt cp b sung. Vo nhng nm 70, bng nhng phng php khc nhau, ngi ta to c nhng phn t DNA ti t hp u tin. Nm 1972, nhm nghin cu ca trng i hc Stranford (Paul-Berg) a ra phng php ui ni cc phn t DNA khc nhau. to u dnh, ngi ta s dng enzyme terminal transferase. Di tc dng ca enzyme ny, ngi ta to c nhng ui poly nucleotide khong 50 n 100 gc adenine (polyA) u OH (3) ca mch n DNA SV 40 (Simian virus 40) v ui polyT DNA plasmid c m bi enzyme. Sau khi trn ln hai loi DNA ny, cc ui b sung adinine v thymine bt cp li vi nhau v vi s c mt ca enzyme ligase, hai DNA ny c ni li v to ra plasmid ti t hp vng. V s an ton cho cng ng nn sn phm ti t hp ny khng c bin np trong t bo ch. Tuy cha hon tt, nhng thc nghim ca Berg cho ta hai vn mu cht v DNA ti t hp. ng cho rng, c th dng enzyme ct DNA mt cch nh trc v cc on DNA cc loi khc nhau c th ni li vi nhau. Nm 1973, Staley Cohen v Annie Chang to ra nhng phn t DNA ti t hp t cc loi khc nhau, c nhiu u im v c bin np trong t bo ch. V t y, cng ngh DNA ti t hp ra i. Sau , nhiu nh khoa hc lao vo cc th nghim lp ghp gen v nhanh chng thu c nhng kt qu c ng dng trong thc tin. K thut ti t hp DNA do Rerg, Chang, Boyer v Cohen ra gip cc nh sinh hc c th lm thay i tnh di truyn ca c th sng theo chiu hng nh trc v vt qua c hng ro ngn cn loi. Mi t hp DNA mi u to nn nhng c im sinh hc mi k c v mt di truyn ln sinh ha.

Vy ngi ta gi DNA ti t hp l mt DNA lai, tm c invitro (trong ng nghim) bng cch t hp 2 DNA thuc 2 loi khc nhau. V d: Hai vector l plasmid, phage c ci mnh DNA l to ra DNA ti t hp (Hnh 6-2). Cc DNA l ny c gi l on ci hay DNA ngoi lai. N chnh l mt mnh (on) DNA hay l gen m ngi nghin cu quan tm v ghp n vo DNA ca plasmid hay DNA ca virus.
DNA plasmid DNA l DNA phage
Vector
Plasmid Phage DNA l
Hnh 6-2: M hnh biu din s to ra DNA ti t hp 6.1.1.2- Mc ch to DNA ti t hp DNA ti t hp c ch tc vi mc ch l thc hin mt qu trnh gi l s tch dng. Tch dng l s tch lp v thu nhn nhiu bn sao ng nht ca mt gen hay ca mt mnh on DNA. Tch dng c th c i km hoc khng c i km vi s biu hin protein. Nhng p dng ny ca DNA ti t hp dng thit lp cc ngn hng b gen, cDNA hoc tng hp protein, enzyme, hormone, ... 6.1.1.3- Nhng yu cu ca DNA ti t hp - DNA ti t hp phi t c nhng yu cu cn thit ca mt vector chuyn gen (xem Chng 4). - DNA ti t hp phi c hot tnh khi a vo t bo ch.

- DNA ti t hp phi c nhng du hiu d pht hin trong qun th vi khun. V d quan st s hot ng v biu hin ca gen ti t hp. 6.1.2- Cc cng on chnh to DNA ti t hp 6.1.2.1- Chn nguyn liu DNA c chn lm phng tin vn chuyn gen (vector) thng l DNA plasmid v DNA phage. Chng c thu nhn ch yu t t bo vi sinh vt, c tch v lm sch theo phng php chit tch DNA plasmid, DNA virus (Chng 7). DNA cha gen cn nghin cu (DNA l) cng c thu nhn t t bo sinh vt c chit tch v lm sch v kim tra theo phng php chit tch DNA. Ngoi ra, ngi ta cn s dng DNA tng hp bng phng php ha hc hoc tng hp t mRNA nh enzyme sao chp ngc theo c ch nuclease S1 hoc k thut Gubler-Hoffman. Cc enzyme ct, ni cc on DNA li vi nhau nh enzyme ristriction, enzyme ligase cng vi s hp tc ca mt s enzyme khc nh kinase v alkanline phosphotase, ... 6.1.2.2 Cng on ct vi s tham gia ca enzyme RE kiu II RE (Restriction Endonuclease) kiu II: l nhng enzyme ct hn ch nhng v tr xc nh v c dng trong cng ngh DNA ti t hp bi v chng c mt s u im: Hu nh ch c mt kiu hot tnh ct, enzyme khc m nhim vic ci bin. Mi mt enzyme ct v tr ph hp nh trc ngay bn trong hay cnh trnh t nhn bit. Chng ch cn ion Mg v khng cn nng lng ATP. V d: EcoRI:
G AATTC CTTAA G vt ct
+2
Bam HI:
G GATCC CCTAG G vt ct

- Cc enzyme ny gp chui ch ca n trn phn t DNA v ct DNA thnh hai mnh dng u bng hay u dnh. Hiu qu ct ca enzyme gii hn ph thuc vo nhiu yu t khc nhau nh hm lng ca enzyme, nhit , ion kim loi, mi trng m, pH v tinh khit ca DNA. 6.1.2.3- Cng on ni vi s c mt ca DNA ligase Bc tip theo ca k thut di truyn l ni cc on DNA vo vector chuyn gen to plasmid c mang DNA l. Phn ng ni c thc hin nh enzyme DNA ligase. DNA ligase l mt enzyme xc tc cc phn ng ni hai mnh DNA bng cch to cu ni phosphodiester gia u 5(P) v u 3(OH) ca hai nucleotide ng cnh nhau (Hnh 6-2). Trong sinh hc phn t, ngi ta coi DNA ligase nh mt cht keo phn t kt dnh cc mu DNA li vi nhau. 1,- Ni u bng: Ni u bng c xy ra khi hai on DNA u bng ng cnh nhau. Di tc dng ca enzyme ligase, lin kt phosphodiester gia u 5(P) v u 3(OH) c hnh thnh v hai nucleotide c ni li vi nhau (Hnh 63a). Kh nng ni hai on DNA u bng rt thp. tng hiu sut phn ng, thng ngi ta phi tng nng ca cc on DNA. 2,- Ni u lch: u tin, hai mnh DNA u lch do c nhng baz b sung nn chng tin gn li nhau to lin kt hydro gia cc baz nit b sung. Sau , hai nucleotide ca hai u ni to lin kt este gia OH(3) v P(5) di tc dng ca enzyme DNA ligase. Phn ng ni c thc hin theo s c biu din trn Hnh 6-3b.
a,- Ni u bng:
OH P 5
C C G G
+
5 P 3 OH
A T T A
OH P
C C G G
A T T A
P OH
DNA ligase
C C A T G G T A
b,- Ni u lch:
G C C T A G G A T C T A
Bt cp baz b sung
P OH 3 5
G G A T C T C C T A G A
G A T C T G C C T A G
3 P
DNA ligase
A
5 OH
Hnh 6-3: DNA v cc phn ng ni

6.1.3- Cc phng php to DNA ti t hp 6.1.3.1- Phng php n gin dng u lch 1,- Chn v x l DNA plasmid: Plasmid c tch t t bo E. Coli hoc t bo nm men theo phng php chit tch DNA plasmid. Sau , dng enzyme RE II (nh EcoRI) ct to ra plasmid h c hai u lch nhau. 2,- Chn v x l on ci: DNA on ci c thu nhn v la chn, tinh ch theo mc ch ca tng nghin cu. Ct DNA bng enzyme RE loi II cng loi vi enzyme ct plasmid (EcoRI) to ra cc vt ct ging nhau. 3,- To plasmid ti t hp: Ghp on ci vo plasmid bng cch cc DNA on ci vi plasmid vi s c mt ca enzyme DNA ligase, ta thu c DNA ti t hp. Phn ng ni xy ra theo s c biu din trn Hnh 6-4. 6.1.3.2- Phng php s dng on ni (linker) 1,- Chn v x l vector plasmid: (tng t nh trn) 2,- Chn v x l on ci: Linker l nhng on DNA di 10 n 15 nucleotide, c tng hp bng phng php ha hc, sao cho gia mi linker phi c trnh t nhn bit bi mt enzyme ct hn ch loi II. V d nh EcoRI c chui ch l G/AATTC. cDNA u bng c tng hp t mRNA nh enzyme sao chp ngc theo c ch nuclease S1. Metyl ha cc vng hn ch c trong on cDNA si i c nhn bit bi enzyme EcoRI. linker c cha chui ch G/AATTC vi cDNA v enzyme DNA ligase to phn t lai. Ct phn t DNA lai bi enzyme EcoRI, ta c phn t lai c hai u lch.
DNA l
Ct bi Eco RI (REH ) Aatt Ct bi Eco RI (REH ) TtAa TtAa Aatt
Aa
tt
Hnh 6-4: Phng php dng u lch
Tt aa
Tt aa
Aatt TtAa
DNA ligase
a Tt
DnA ti t hp
tt Aa
a
aa Tt

im nhn bit ca Eco RI C C G A A T T C GG GG C T T A A G C C
Linker Cc phn t linker
DNA l (cDNA)
DNA ligase
DNA plasmid
Eco RI
Eco RI
DnA ti t hp
Hnh 6-5: Dng cc on ni linker to DNA ti t hp

3,- To vector ti t hp: to vector ti t hp, ngi ta ghp phn t DNA lai u lch vo plasmid m cng c ct bi cng mt enzyme EcoRI. Nh tc dng ca enzyme DNA ligase m vector ti t hp c to thnh (Hnh 6-5). 6.1.3.3- Phng php s dng enzyme terminal transferase (ETT) Nguyn tc: Da vo c tnh ca enzyme terminal transferase c kh nng gn cng mt loi nucleotide vo u 3(OH) ca phn t DNA. Cch to DNA ti t hp bng phng php ny c tin hnh theo 4 bc sau (Hnh 6-6): 1,- Bc 1: Chn v phn lp DNA l u bng v plasmid, gi s ta phn lp plasmid c cha chui ch c nhn bit bi enzyme BamHI (G/GATCC). Ct plasmid bng enzyme BamHI to plasmid h c hai u dnh. Ct DNA l bng enzyme exonuclease theo hng 5 3 to DNA c hai u lch nhau. 2,- Bc 2: DNA l vi cng mt loi nucleotide dCTP vi s c mt ca enzyme terminal transferase to ui polyC u 3(OH) ca DNA l. plasmid vi cng mt loi nucleotide dGTP vi s c mt ca enzyme terminal transferase to ui polyG u 3(OH) ca plasmid h. 3,- Bc 3: a DNA l vo plasmid vi s c mt ca enzyme DNA ligase, cc u mt ca homopolymer c trnh t b sung (---GGGG 3/3CCCC---) s bt cp vi nhau. 4,- Bc 4: B sung enzyme DNA-polymerase I gn cc nucleotide tng ng vo ch trng theo nguyn tc b sung. Enzyme ligase ni lin kt

phosphodiester v cui cng to c plasmid ti t hp c hai chui ch c nhn bit bi enzyme BamHI. u im ca phng php l ghp DNA l u bng vo plasmid to DNA ti t hp v ch tc c DNA u lch t DNA u bng.
3 5 5 3 Ct DNA bng E exonuclease 5 3 3 5 PolyC ETT CCC CCC 5 3
GGA T C C C C T A GG
Plasmid cha chui ch ca BamHI
Ct bng E BamHI (3) GA T C C G G C C T A G (3)
PolyG ETT (3) GGGGGA T C C G C C T A GGGGG (3) G
CCC G C C T A GGGGG
GGGGGA T C C CCC G
BamHI GGA T C C C C C C C T A G G GGG
BamHI GGGG G A T C C C C C C C T A GG DNA ti t hp
Linker
Linker
DNA l
Hnh 6-6: Cch to DNA ti t hp dng enzyme terminal transferase
6.2- MT S PHNG PHP A DNA TI T HP VO T BO CH 6.2.1- Ha bin np Hin tng bin np l cha kha gip ta hiu bit c s phn t ca gen, cng l cng c thc hin cc thao tc to tnh di truyn ca vt sng. Theo Mandel v Higa cho thy rng, E. Coli tr nn rt d b bin np bi DNA ngoi lai khi cc t bo vi khun c x l trong mi trng c CaCl2 v trc c sc nhit 42C. Ha bin np l phng php s dng cht ha hc, to iu kin a vector ti t hp vo t bo ch. Qu trnh c thc hin theo hai buc sau: X l t bo ch trong dung dch CaCl2 nhit thp nhm thay i mng t bo v vector ti t hp vi t bo ch x l. Hiu sut ca phng php ha bin np ny vo khong 10 n 10 t bo bin np trn 1mg DNA ti t hp. Qua cc kt qu thc nghim, ngi ta thy rng, cc t bo pht trin pha sm n pha gia d c bin np hn. Nhng nghin cu sau ny cho thy vic x l t bo bng cc ion kim loi ha tr hai nh Mg , Mn v Ba cng cho kh nng bin np ln. Ngoi ra, hiu sut bin np cn ph thuc vo kch thc ca plasmid, plasmid cng nh th hiu sut bin np cng cao. 6.2.2- in bin np Nguyn tc: S dng dng in cao th cc b theo xung to l nh trn mng sinh hc ca t bo, to iu kin cho t bo hp th DNA ti t hp c d dng. Hiu sut: T 10 n 10 t bo bin np cho 1mg DNA ti t hp, tuy nhin, lng t bo bin np b cht nhiu c khi ln ti 70%. Hiu sut ca phng php ny ph thuc vo cc yu t sau: - mnh ca in trng tc ng khc nhau i vi cc loi t bo khc nhau,
9 10 +2 +2 +2 5 6

- di ca hng s thi gian (thi gian ngt xung - ms). Theo nhiu nghin cu, hu ht i vi cc loi t bo sinh vt, hiu qu bin np cao khi hng s thi gian t c khong 6ms, - Nng t bo ch, - Nng DNA ti t hp, - Giai on pht trin ca t bo (t bo qu gi hoc qu non u khng thch hp), - Mi trng dung dch m, - Cu trc mng t bo. 6.2.3- Bin np t bo trn (protoplast) L phng php chuyn DNA ti t hp vo t bo ch c x l bng polyetylen glycol (PEG). to t bo trn, ngi ta t bo vi PEG nng 30% n 40%. y l phng php chuyn gen c hiu qu cao i vi t bo thc vt. Bng phng php ny, ngi ta nhn c cc cy mang gen bin np n nh v di truyn qua nhiu th h (Potrykus v cng s - 1995). u im: Tn s bin np ng thi gen ch th v gen cn bin np cao. C th chuyn gen vo t bo protoplast ca bt k loi cy no. c bit l loi cy c gi tr kinh t cao nh la, ng, i mch. Nhc im: Vic ti sinh cy protoplast cn rt kh khn mt s loi cy. 6.2.4- Phng php bn gen Nguyn tc: Ngi ta s dng ht kim loi nng c bao bc DNA v bn trc tip vo t bo. u im: Phng php ny c th bin np cho tt c cc loi t bo thc vt. Thao tc d dng, bn mt ln c nhiu t bo. Nhc im: Hiu sut bin np thp, thng xuyn nhn c cy bin np khm (cy c t bo bin np v t bo khng bin np).

Mt s thnh tu t c bng phng php bn gen: Nm 1988, Mc. Cabe v cng s nhn c cy u tng bin np u tin bng phng php bn gen. Nm 1990, Promm, Gordon, Kamm v cng s nhn c cy ng bin np nhiu phng th nghim. Nhng nm gn y c hng lot cng b v bin np thnh cng la. Nm 1996, Zthang v cng s bin np u , ma v bng. iu khng nh tnh u vit ca phng php ny. 6.2.5- Phng php vi tim Phng php vi tim l phng php s dng vi kim v knh hin vi a DNA ti t hp vo mi t bo nht nh. y l qu trnh lai ghp cho t bo bc cao hoc t bo hp t. Ty thuc tng trng hp c th, ngi ta c th chn phng php sao cho vic a DNA ti t hp vo t bo c hiu qu cao. u im: C th ti u lng DNA ti t hp a vo t bo v quyt nh a DNA vo loi t bo no. a chnh xc v thm ch vo tn nhn ca t bo v c th quan st c. Cc t bo c cu trc nh nh ht phn, t bo tin phi cng c th tin hnh mt cch chnh xc. C th bin np cho mi ging cy. Nhc im: Mt pht tim ch c mt t bo v thao tc cn phi kho lo v t m. 6.2.6- Ti np Ti np l hin tng chuyn vt liu di truyn qua vector l virus t vi khun cho sang vi khun nhn, trong c qu trnh chuyn gen v ti t hp gen vi khun nh thc khun th (Bacteriophage). Thc nghim chng minh c ti np E. Coli qua phage , phage P1 v Bacillus subtilis qua phage SP10 . So vi cc phng php trn, ti np cho hiu sut cao hn. Nh vy, n nay c nhiu phng php ha hc, ha l, c hc v sinh hc a DNA ti t hp vo t bo ch. Ty cc i tng v yu cu c th, phng php ny hay phng php khc c hiu qu v c s dng nhiu hn.

6.3- K THUT TCH DNG (to dng) K thut tch dng bao gm vic phi ci mt mnh (chui) DNA l vo mt vector (plasmide hoc phage ) bng phng php ha sinh. Sau , a phn t lai ny vo t bo ch chn la bng phng php bin np hoc ti np. Trng hp mun to dng tng hp enzyme th mnh DNA nh ci phi m ha cho gen cu trc ca mt enzyme no . 6.3.1- Mc ch ca s tch dng - Thit lp ngn hng b gen, - Thit lp ngn hng cDNA, - Sn xut protein, enzyme, - Sn xut vaccine, - Sn xut khng sinh. 6.3.2- Cc bc c bn ca phng php tch dng Qu trnh thc hin c th thay i ph thuc vo nhn t tham gia v mc ch ca qu trnh tch dng. Tuy nhin to dng, cn phi thc hin cc bc sau: 6.3.2.1- Tch lp cc DNA l cn to dng Chn v ct DNA l ca t bo cho bng mt enzyme ct hn ch (RE). Phn lp on DNA (gen qu) ph hp vi vector v mc ch cn to dng. Trong trng hp c bit, to dng mt gen cha bit, ngi nghin cu c th tng hp ha hc on DNA cn to dng khi d on cu trc protein do gen ch huy tng hp hoc tng hp cDNA t mRNA. To u dnh khi cn thit. 6.3.2.2- Chn v x l vector Chn vector cn phi ch nhng yu cu sau: ln ca gen l (on ci), loi t bo ch tip nhn vector v phng php ng dng.

X l vector: ct vector bng enzyme ct hn ch cng loi vi enzyme ct DNA ni trn ( to nhng vt ct ging nhau, thun tin cho vic ni ghp sau ny). Kh nhm phosphat bng enzyme alkanline phosphotase trnh hai u vector ng kn tr li. 6.3.2.3- To DNA ti t hp (Vector ti t hp) Vic to DNA ti t hp bng cch ghp DNA l vo vector c ct cng mt enzyme ct hn ch loi II, khi , chng s ghp i nhng u dnh li vi nhau nh bt cp b sung. Mt phn ng ghp ni xy ra vi s c mt ca enzyme DNA ligase ca E. Coli hoc phage T4 hon chnh phn t lai. 6.3.2.4- Chuyn DNA ti t hp vo t bo ch bng phng php bin np hoc ti np Cng on ny nhm mc ch s dng b my ca t bo ch sao chp vector ti t hp thnh mt s lng ln bn sao. Vic chuyn DNA ti t hp vo t bo vi khun tc l lm cho vi khun tr thnh kh bin, ngha l c kh nng thm vector ti t hp. S thm ny c th xy ra mt cch t nhin hoc c cm ng. Tuy nhin, n s ph thuc vo loi plasmid s dng lm vector v ph thuc vo s nh v ca vng ci lp cha bn trong vector m ngi nghin cu s chn phng php bin np hoc ti np. Bin np l hin tng chuyn vt cht di truyn trc tip t t bo th cho D (Doner) sang t bo th nhn R (Reception), khng cn s tip xc gia hai t bo hoc nhn t trung gian l phage hoc virus. Bin np c thc hin vi vector chuyn gen l plasmid. C nhiu phng php bin np nh ha bin np, in bin np, bin np t bo trn, phng php bn gen v phng php vi tim. Ti np l hin tng chuyn vt cht di truyn trc tip t t bo th cho D (Doner) sang t bo th nhn R (Reception) qua nhn t trung gian l virus. Ti np c thc hin vi vector chuyn gen c ngun gc l virus nh phage, cosmid, ...

6.3.2.5- Pht hin dng cn tm Cng vic tip theo l kim tra s hin din ca gen mong mun. Vic chn la nhng chng nh mun l khng n gin, v cch tin hnh th nghim trong mt hn hp khng ng nht nn cc dng vi khun c th mc ln theo ba kh nng: - T bo vi khun khng nhn plasmid ti t hp, - T bo nhn plasmid nhng khng c gen l, - T bo vi khun nhn c plasmid ti t hp. Ty thuc vo mc ch nghin cu m ngi ta c cc phng php khc nhau xc nh dng cn tm. Nu mc ch l nghin cu on gen cha bit, ngi ta thng dng u d. Nu on DNA bit (cDNA), cng vic s n gin v nhanh chng. 6.3.2.6- Kim tra v thu nhn sn phm ca gen ti t hp Ty tng trng hp c th m ngi nghin cu a ra nhng phng php kim tra thu hi sn phm ca gen ti t hp. Nu l mc ch thit lp ngn hng cDNA, ta phi tin hnh cc bc sau: u tin ngi ta phi tch plasmid ti t hp ra khi dch nui cy, sau , ct plasmid ti t hp bng enzyme RE loi II v thu c hai bng DNA, mt c di bng khung vector, cn bng khc c di tng ng ng bng cDNA. Cch kim tra li cDNA c ct bng cch in di trn gel agaroza 0,8% v kim tra li di ca nhng bng cDNA thu c. Nhng on c kch thc khc nhau s di chuyn nhng khong cch khc nhau trn gel. Nu sn phm ca gen ti t hp l protein, enzyme, hormone, vaccine, khng sinh,... ngi ta c th thu nhn sn phm bng phng php chit tch lng lc ly tm kt ta phn on hoc trao i ion. 6.3.3- Mt s phng php xc nh dng cn tm 6.3.3.1- Phng php lai axit nucleic 1,- Khi nim u d: Cc u d l nhng mnh DNA c nh du, c kh nng nhn bit mt chui DNA hoc RNA ng ng qua lai ha.

2,- c im ca u d: L mt on axit nucleic (DNA hoc RNA) si n. u d phi b sung cc baz nit, i song song vi on axit nucleic cn nhn bit. u d phi so mc c, l cc u nh du phng x. 3,- Cc loi u d: cDNA lm u d cc tt, ngoi ra, mRNA v oligonucleotide cng c th lm u d. Nu chui DNA cha bit trnh t, ngi ta phi tinh sch mt lng nh protein tng ng vi DNA nghin cu v t tng hp u d. Cn nu mt phn DNA phi so mc bit th cng vic rt d dng tng hp mt u d. 4,- Nguyn tc ca phng php lai axit nucleic: Da vo kh nng bin tnh khi nhit tng v hi tnh khi h nhit t t ca DNA. Khi tng nhit ca DNA ln qu nhit sinh l (thng khong 80 n 95C), hai si n DNA s tch ri nhau do lin kt hydro b t. Khi h nhit t t ca khi DNA bin tnh cng vi cc iu kin thch hp khc, cc mch n bt cp tr li. S bt cp ch c th xy ra khi hai trnh t DNA hon ton b sung cho nhau. Tnh c hiu cc ln ca phn ng lai ny cho php bt k trnh t mch n no cng tm gp mch b sung vi n, mc d chng nm trong hng triu cc trnh t DNA v RNA khc nhau. C th dng ng v phng x nh du pht hin on lai. 5,- Cch tin hnh: Chun b mu (u) d c nh du bng ng v phng x hoc ha cht. Bin tnh dng cn tm di dng mt si, sau h nhit t t, cc si n tng ng bt cp vi nhau. S bt cp xy ra gia DNA v DNA, RNA v DNA, RNA v RNA. 6.3.3.2- Phng php s dng khng th 1,- Nguyn tc: Da vo phn ng c trng ca mt s protein v khng th m chng ta c th nhn bit c qua phn ng c trng nh phn ng to mu, phn ng kt ta.

2,- Cch tin hnh: Chn khng th c trng cho protein (thuc th cho protein). Tch protein c biu hin trong qu trnh to dng. protein vi khng th c trng. Sau tin hnh nhn bit thng qua cc du hiu ca phn ng c trng gia protein v khng th. 3,- ng dng: Ch yu s dng trong y hc vi hai yu cu sau: DNA cn nghin cu c gn vo vector phi biu hin protein khi to dng v ng thi, vector to dng phi l vector biu hin (v d nh vector gt11). Protein biu hin cn phi c khng th c trng. 6.3.3.3- Phng php pht hin do mt hot tnh v xen on Nguyn tc: Da vo s mt kh nng khng thuc ca vi khun mang vector ti t hp khi chng c nui cy trong mi trng khng sinh. Phm vi s dng: Phng php ny dng cho vector c t hai gen khng thuc tr ln, v d nh vector pBR322 . Trong plasmid pBR322 c hai gen khng thuc AmP v TetR. Trn cc gen ny c nhng im nhn bit ca cc RE ni ci t DNA l. Khi gn DNA l vo mt trong hai gen ni trn th gen nhn on ci mt kh nng khng thuc. Quan st nhng khun lc b mt gen khng thuc, chng t nhng khun lc c cha gen cn to dng. u im ca phng php l t tn km so vi phng php lai. 6.3.3.4- Phng php pht hin do thay i kiu hnh 1,- Nguyn tc: Da vo s thay i mu sc ca khun lc, chng t rng, khun lc c cha gen l. 2,- Cch tin hnh: Chun b hai mu th nghim m plasmid to dng c cha gen lacZ (v d nh dy pUC), mt mu so snh cn mu kia ci DNA l vo gen lacZ. Sau , nui cy c hai mu trong mi trng c cht cm ng X-Gal (5
R

brom-4chloro-3indolyl D-galactopynoside). Cht cm ng ny b phn hy bi enzyme -galactosidase to ra galactose v X (dn xut indol), ngoi mi trng, dn xut ny b oxy ha cho mu xanh m. Qua quan st, ta thy plasmid khng cha gen l cho khun lc mu xanh do gen lacZ tng hp enzyme -galactosidase. Cn plasmid th 2 cho khun lc mu trng, iu chng t n cha DNA l c ci trong gen lacZ v chn nhng khun lc mu trng. 6.4- NGN HNG GEN (Genomic library) Ngn hng b gen ca mt sinh vt l tp hp cc trnh t DNA cu thnh b gen c gn vo vector, n ch biu din tng s ca gen. Ngn hng ny c to ra t mt b su tp DNA ti t hp c cc chui m ha ln cc chui khng m ha. Khc vi ngn hng cDNA, ngn hng b gen c th c thit lp bt k loi t bo no ca sinh vt nghin cu. Cc bc thit lp ngn hng gen: - Tch v lm sch DNA ca sinh vt, - Ct DNA thnh nhng on c kch thc xc nh bng enzyme RE loi II - thng thng, ngi ta s dng EcoRI, - Vector c chn to dng gen thng l cosmid hoc phage cng c x l bi EcoRI, - To vector ti t hp v ng gi trong v phage, - a vector ti t hp vo t bo ch v to dng, - Xc nh c tnh v biu hin ca dng vi khun va lp, - Tin hnh sng lc, ngha l phi tch nhng vector no c on DNA a chung, t xy dng ngn hng b gen. c im v ng dng: - Ngn hng gen cha cc on DNA ln hn 100 ln so vi cDNA. - Gii m thng tin di truyn cha trong b gen, c bit l cu trc intron v exon ca mt gen xc nh.

- To dng nhng trnh t DNA khng m ha nm cnh cc gen m ha v ng vai tr quyt nh trong s iu ha biu hin ca gen. 6.5- NGN HNG cDNA 6.5.1- Thit k ngn hng cDNA Ngn hng cDNA l tp hp cc bn sao cDNA t tt c cc mDNA ca mt t bo. Nh vy, ngn hng c thit lp t mt loi t bo xc nh (t bo bit ha), v th, cDNA mang tnh t bo rt cao. Qu trnh chun b ngn hng cDNA bao gm cc bc sau y: - Chit tch RNA tng s, ta phi chn nhng t bo c cha nhiu mRNA cn thit v sau , lm sch mRNA, - Chn la cc mRNA cha nhiu A (polyA) bng cch tch trn ct xenlulose oligo dT, - Tng hp cDNA si kp u bng t mRNA di tc dng ca enzyme sao chp ngc nh k thut nuclease S1 hoc k thut Gubler-Hoffman, - Metyl ha cc vng hn ch (c trong on cDNA si i) c nhn bit bi enzyme EcoRI bo v vng ny khng b ct khi thao tc vi EcoRI, - To u dnh bng cch s dng on ni linker c cha trnh t nhn bit bi EcoRI, sau , ct bng enzyme EcoRI. Trong trng hp ny, ch c on ni b ct cn cDNA vn cn nguyn vn (do c metyl ha). a cDNA vo vector to DNA ti t hp, - Ghp cDNA u dnh vo vector thch hp c m EcoRI v kh nhm phosphat, - Hn kn mch nh enzyme phosphoryl ha v enzyme ligase to vector ti t hp, - a vector ti t hp vo t bo ch v nui cy trong cc iu kin thch hp to dng, - Xc nh dng cn tm v thnh lp ngn hng cDNA.

Mc ch ca vic thit lp ngn hng cDNA l nhm nghin cu s biu hin ca mt gen xc nh cng vi nhng vn lin quan n s iu ha biu hin gen cng nh mi tng tc gia cc gen trong mt qu trnh sng. Ch : Khi phn tch kt qu thu c ngn hng cDNA, cn ch tc dng sinh l tng thi im th nghim. 6.5.2- Sng lc ngn hng cDNA Mt phng php ph bin nhm sng lc ngn hng cDNA c bt u t vic to canh trng nui cy cha ng nhiu khun lc t ngn hng cDNA. DNA plasmid t mi m cy vi khun gn vi mnh nh nitroxenlulose (mng lai) s b bin tnh v lai vi mRNA tng s. Mi mRNA khc nhau s c lai trn nitroxelulose vi th t cDNA b sung ca n. Cc mRNA bm vo mng lc c tch ra v dng tng hp protein m n m ha bng h thng dch m invitro. Protein hnh thnh c em phn tch xem c phi l sn phm ca mRNA cn tm hay khng, v t , xc nh vi sinh vt nui cy cha gen mong mun. Sng lc tt c cc m nui cy theo cch trn xc nh dng n th hin gen tm kim.

CHNG VII
MT S PHNG PHP NGHIN CU TRONG CNG NGH DI TRUYN 7.1- CC PHNG PHP TCH CHIT AXIT NUCLEIC Mi nghin cu v ng dng ca cng ngh gen u c bt u t vic thu nhn mt lng axit nucleic tinh sch. Mi quan tn u tin ca k thut tch chit axit nucleic l thu nhn c cc phn t ny trng thi nguyn vn, khng b phn hy bi tc nhn c hc, tc nhn ha hc v enzyme nh RNase, DNase. 7.1.1- Phng php tch chit DNA vi khun DNA c kch thc ln, mi vic chit tch cn phi trnh mi tc nhn c hc, ho hc ti phn t DNA ny. Vic tch DNA t t bo vi khun c chia lm bn bc c bn: 7.1.1.1- Nui cy v thu sinh khi Ty tng loi vi khun m ngi nghin cu chun b mi trng v iu kin nui cy khc nhau nh nhit , pH, v iu kin dinh dng thch hp cho s pht trin ca vi khun. Tch t bo ra khi dch nui cy bng cch ly tm v lm sch t bo. Thng thng khong 1.000ml dch nui cy li tm v thu ly khong 10ml cn vi khun. 7.1.1.2- Ph v mng t bo v gii phng cc thnh phn bn trong Axt nucleic c gii phng ra khi mng t bo bng phng php ha hc v sinh hc thu dch chit t bo. Enzyme lysozyme l mt enzyme c trong lng trng trng hoc phage T4, c kh nng ph v thnh phn trng hp ca thnh t bo. EDTA (TrilonB) to phc vi Mg dng ha tan v lm v thnh t bo. ng thi EDTA ngn khng cho enzyme trong t bo phn hy DNA. Ngi ta cn c
+2

th ph v mng t bo v gii phng DNA ra khi lin kt histon bng cht ty ra SDS (Sodium Derecyl Sylfat) hoc enzyme protease. Cc cht ph v mng ny lm li gii mng, tch ri cc phn t lipit v gy ra s gy mng t bo. 7.1.1.3- Lm sch DNA t dch chit t bo Loi b cc thnh phn khng mong mun nh protein, lipit v cc thnh phn khc bng kt ta phn on. Mu c lc mnh trong h dung mi phenol-chloroform bin tnh, kt ta protein v gii phng axt nucleic vo dung dch lng. Protein b bin tnh s tch ra khi pha nc c cha DNA s c tch ra nh ly tm. Dch ni gm axit nucleic v nc c ht ra v tip tc phn hy RNA bng enzyme RNase. Sau , tch DNA v nc thc hin bc phn tch tip theo. 7.1.1.4- Thu hi DNA dng c Mc ch ca bc ny l thu DNA dng c c, nhm bo v chng khi s phn hy ca enzyme. C 2 phng php kt ta: Kt ta DNA trong etanol vi s c mt Na+ nng cao, nhit thp, t l gia etanol v mu phn tch l 3:1. Kt ta trong izopropanol, t l gia ru v mu l 1:1. Vi phng php ny khng cn s c mt ca mui v loi c DNA c phn t lng thp. Ly tm cao tc v thu nhn DNA dng c c. Trong c hai phng php, DNA thu c s ra trong etanol-70% loi izopropanol v mui thu c DNA tinh khit. 7.1.2- Phng php tch DNA plasmid Vic tch DNA plasmid ra khi DNA nhim sc th l rt kh khn. Tuy nhin ngi ta c th da vo s khc nhau l ha gia hai loi DNA nh: kch thc, hnh thi, cu trc ca chng. V nguyn tc c tin hnh theo 3 bc sau:

7.1.2.1- Bc 1 Sau khi thu hi v lm sch cc t bo c cha plasmid, ngi ta x l trong hn hp SDS, EDTA hoc NaOH lm DNA si i bin tnh thnh DNA si n nm cnh nhau. 7.1.2.2- Bc 2 X l hn hp trn trong mi trng m (NaCH3COO+CH3COOH) kt ta protein v SDS dng tp hp. 7.1.2.3- Bc 3 a v mi trng trung tnh, DNA si n c hi tnh tr li. Cn DNA (NST) do di nn hi tnh chm v kt ta cng SDS. Tch DNA plasmid bng cch kt ta trong etanol hoc izopropanol, sau ly tm tch c plasmid tinh sch. 7.1.3- Tch DNA ca t bo khc Ngoi vi khun, DNA ca virus, ca t bo thc vt, ng vt cng c th tin hnh tch phc v cho cng ngh di truyn. Cc giai on lm sch tng t nh trn. Ch c giai on nui cy v ph v mng t bo th khc, tuy nhin, ty tng loi t bo m c nhng cch x l sao cho thch hp. 7.1.4- Phng php tch RNA tng s v mRNA 7.1.4.1. Tch RNA tng s RNA c tin hnh chit tch gm cc bc c bn tng t nh DNA (bao gm ph v mng t bo, loi protein, ta RNA) nhng y ta dng enzyme desoxyribonuclease (DNase) phn hu DNA. Do RNA km bn v d b phn hu bi enzyme ribonuclease (RNase) c nhiu khp ni, hot tnh cao li bn vi nhit (trn 90C), v vy, iu kin thao tc chit tch phi c tin hnh trong mi trng v cng nghim ngt. Phng php chung tch RNA tng s c tin hnh nh sau: T bo c nghin vi cht ty ra SDS, sarcocyl nng cao, nit lng bin tnh protein. ng thi mu cng c nghin vi cht kh (2-

mercaptoethanol) c ch RNase. Nc s dng cng c x l vi DEPC (Diethylpyrocarbonat) cng loi b RNase. Cc protein, DNA c loi b qua x l mu vi hn hp dung mi Trizol (phenol pH=4, guanidinium thiocyanate, glycerol, sodium acetate), khi ly tm, ta tch c RNA trong pha nc. DNA pH=4 b hp ph vo pha di cng vi phenol, protein bin tnh nm gia hai pha cng b loi cng vi DNA cng vi pha phenol. RNA trong pha nc c ht ra sau ta bng izopropanol -20C, li tm v ra li bng ethalnol 79%, thu hi RNA tinh khit, bo qun lnh lm cc th nghim tip theo. Cht lng ca RNA tng s c nh gi s b qua in di trn gel agarose. 7.1.4.2. Tch tng loi RNA khc nhau Da vo kch thc trng lng phn t, ngi ta c th tin hnh sc k, in di hoc ly tm tch tng loi RNA. Phng php tch mRNA: Do kch thc b li chim lng nh (15%) RNA tng s, c cu trc a dng nn khng th tch bng phng php trn. Nh c im mRNA c ui polyA, do , ngi ta tch mRNA ra khi mu bng phng php sc k i lc oligodt-xenlulose. Ngy nay trn th trng c loi vin bi t, trn b mt c gn oligodt. Sau khi mRNA bm trn b mt cc vin bi t, thng qua lin kt b sung vi oligodt, cc vin bi ny thu nhn v em ly tm ta thu c mRNA tinh khit. Lu rng, tch mRNA, ngi ta i t t bo bit ho, lng mRNA nhiu v y cng l con ng nghin cu cu trc gen m ho. Ngy nay ngi ta tng hp c ngn hng mRNA. 7.1.5- Tch v thu nhn gen Ngy nay, ngi ta c th thu nhn gen thc hin k thut di truyn bng ba phng php: 7.1.5.1- Tch cc on DNA t b gen y l phng php s dng rng ri ngay t bui u tin ca s pht trin k thut DNA ti t hp. Ton b phn t DNA ca mt sinh vt c ct nh thnh nhng on c kch thc 1,52kb bng restriction enzyme

(RE). in di hoc sc k tch on DNA tinh khit, sau gn vo vector chuyn gen, to plasmid DNA ti t hp. Nhc im l tn nhiu cng sc v thi gian nhng ngy nay ngi ta vn s dng to ngn hng DNA b gen (genonic DNA libraries). 7.1.5.2- S tng hp gen bng phng php ho hc Nm 1969, gen nhn to u tin c tng hp do nhm nghin cu Khorama, l gen m ho tng hp tRNA vn chuyn amino axit (alamin) nm men gm 77 cp nucleotide. Gen u tin khng c biu hin v khng c cc trnh t iu ho. V sau, chnh nhm ny tng hp c gen c hot tnh: l gen m ha cho cht c ch tRNA vn chuyn tyrosine E. Coli, c chiu di khong 200 cp nucleotide. Vo nm 1977, K. Itakura v Boyer tng hp c gen m ha sinh tng hp hormone somatostatin ca ng vt c v c biu hin E. Coli. v t , ni E. Coli mang gen tng hp ha hc c to ra. Sau ny, ngi ta tng hp ha hc gen m ha hormone tng trng ca ngi d 584pb. Phng php tng hp ha hc gen cng c s dng to ra ni vi khun tng hp insulin, mt loi hormone cha bnh tiu ng cho ngi. Gen insuline c tng hp t hn 40 on oligonucleotide khc nhau, sau , c ni li nh enzyme lygase to ra hai chui polynucleotide di 271 v 286bp m ha, tng polypeptide A c 21 aminoaxit, polypeptide B c 30 aminoaxit. Qua cc kt qu thu c, cc nh khoa hc khng nh rng: mun tng hp c gen bng phng php ho hc cn phi bit trnh t ca axit amin protein m gen chu trch nhim tng hp. 7.1.5.3- Sinh tng hp gen t mRNA Tng hp cDNA t mRNA nh k thut ca Gubler v Hoffman vi s c mt ca enzyme phin m ngc (Rever transcriptase - Hnh 7-1). Sau , cDNA (complementary) gn vo plasmid v bin np vo vi khun to dng cDNA. Phng php ny c trnh by Chng 6 (thit lp th vin cDNA).

u im l: Gen thu nhn bng phng php ny loi b c nhng trnh t khng m ho. Bng con ng ny, ngi ta to dng gen m ho tng hp globine ca ngi, ng vt v chim, protein thy tinh th mt b, ovalbumine (protein lng trng trng) v fibroin t tm. Vo nm 1992 cc nh khoa hc M to c dng cDNA ca 2.375 gen ca b no ngi. Phng php tng hp gen t mRNA ngy cng c pht trin, n kt hp vi cc phng php khc ca sinh hc phn t v c ng dng trong nhiu lnh vc.
5 Enzyme - RT Cu trc kp tc NaOH, R Nase 3
m-RNA
AAAA TTTT-mi oligo-dT
DNA -polymerase cDNA Nuclease S1
cDNA si kp
Hnh 7-1: S tng hp gen t mRNA 7.2- PHNG PHP NHN BN (PCR - Polymerase Chain Reaction) Phng php to dng invivo m chng ta cp trong Chng 6 tuy gii quyt v vn s lng, nhng i hi thao tc qu phc tp v thi gian qu di. Trong s ra i ca cc phng php khuch i (ti bn nhanh) invitro c chn lc, chng ta phi k n phng php PCR. Phng php PCR - phn ng dy chuyn nh hot ng ca enzyme DNA-polymerase - do K. Mulis cng cng s pht minh nm 1985, a li mt cuc cch mng trong di truyn hc phn t. y l phng php hon ton mi trong vic nghin cu, phn tch gen v h gen. Kh khn ln nht trc y trong vic phn tch gen l ch chng l nhng mc tiu n l v

rt nh trong mt h gen phc tp khng l. K thut PCR ra i thay i tt c, gip chng ta c th to ra mt s lng ln cc bn sao ca mt on DNA mong mun. Do nhng u im tuyt i trong nghin cu sinh hc phn t, k thut PCR c nhanh chng p dng rng ri chun on cc bnh v virus, vi khun, cc bnh k sinh trng v cho kt qu rt chnh xc. Mt khc, s phn tch thnh phn v trt t nucleotide trn phn t DNA trong h gen cn c ngha to ln trong phn loi cc loi sinh vt. Chnh nh tnh thc tin to ln ca k thut ny m tc gi ca PCR, Kary Mulis, c tng gii thng Nobel vo nm 1993. Thc cht y l phng php to dng invitro, khng cn n s hin din ca t bo v da trn nguyn tc c trnh by sau y: 7.2.1- Nguyn tc ca phng php PCR Nguyn tc ca phng php l to lng ln cc on DNA c th t DNA khun da trn c s hot ng ca DNA-polymerase tng hp si mi b sung. Cc yu t c bn thc hin phn ng PCR bao gm: - Si khun DNA ch cn bit trnh t nucleotide ca on nh nm cnh on cn nhn thit k hai mi oligonucleotide. - Hai on mi ngn xc nh cc im bt u tng hp DNA. L tn hiu ch hng i (5 3) ca enzyme DNA-polymerase. Mi di khong 20 nucleotide v cc nucleotide hai u ca mi khng t kt hp vi nhau theo nguyn tc b sung. - C y cc loi nucleotide dATP, dTTP, dGTP, dCTP. - Mi trng m cung cp ion Mg v nc tinh khit khng c enzyme RNase v DNase. - Enzyme chu nhit Thermus aquaticus (Taq) Dung tch tng s cho mt phn ng PCR khong t 20 l n 50l. c im ca phn ng PCR l chn lc, nhy v nhanh.

7.2.2- Cc bc tin hnh th nghim Phn ng PCR l mt chui gm nhiu chu k v mi chu k gm 3 bc (Hnh 7- 2 v Hnh 7-3):
3 5 Bin tnh 3 5 B sung mi 3 5 3 B sung DNA -polymerase 3 5 Trnh t mc tiu 5 3 3
3 5
5 3
Hnh 7-2: Cc bc thc hin phn ng PCR mt chu k 7.2.2.1- Bc 1 - Thc hin qa trnh bin tnh DNA bng nhit a DNA vo dung dch phn ng (gm cc thnh phn cn thit cho s sao chp), tng nhit ca dung dch ln ti 9098C, thi gian lu tng ng khong t 30s1 pht. Ti nhit ny, cc phn t DNA mch kp b tch ra (do lin kt hydr b t), to nn cc si n dng lm khun tng hp si mi.

7.2.2.2- Bc 2 - Thc hin phn ng lai Sau bc 1, ngay lp tc nhit c h xung t t v nh hn nhit Tm ca mi, vo khong 3768C v thi gian lu 30s1 pht. B sung mi mi bt cp vi si khun. Mi c tng hp ha hc, c mi ngc v xui. Ngi ta cn c th da vo trnh t nucleotide u 3 ca khun tng hp mi. Sau b sung DNA-polymerase ko di mi. 7.2.2.3- Bc 3 - Tng hp mch mi hay cn gi l ko di (extension) Nng nhit phn ng ln 72C trong vi chc giy n 1 pht DNApolymerase tng hp si mi. Trong thc t, thi gian v nhit phn ng ph thuc vo si DNA cn khuch i. Kt thc mt chu k t mt DNA kp m tng hp 2 si DNA kp con. C nh vy, phn ng xy ra trong 25 n 40 chu k. Sau chu k cui, nhit c duy tr 72C trong khong t 5 n 10 pht sao cho tt c cc si n xon li v to nn sn phm ca PCR. Sau h xung 4C bo qun sn phm. Sn phm ca PCR c kim tra bng cch chy in di trn gel agarose nng t 0,8%2% pht hin s a hnh ca cc on DNA c th, hoc cc on DNA b thay i do cc tc nhn no (t bin, ti t hp).

Bin tnh Khun DNA B sung mi
Chu k 1
Ko di
Bin tnh
B sung mi
Chu k 2
Ko di
Bin tnh
B sung mi
Chu k 3
Chu k 4
Bin tnh B sung mi Ko di
Chu k 5
Bin tnh B sung mi Ko di
Hnh 7-3: S phn ng PCR nhiu chu k

7.2.3- Cc yu t nh hng n phn ng PCR 7.2.3.1- DNA mu lm khun Phn ng PCR ti u xy ra khi mu DNA khng c di qu, thng khuch i tt nht i vi on DNA di khong 11,5kb. Mu DNA tinh sch s cho kt qu tt, tuy nhin, k thut chun on bng phng php ny vn t kt qu tt trong trng hp mu DNA khng cn c tinh sch cao, nh vt mu kh, nhng mu vt kho c (tc, mng tay ngi cht). Phng php ny cho php xc nh DNA vi mt lng thp, khong nh hn 100 ng (1g = 109ng). 7.3.3.2- Enzyme DNA-polymerase Cht lng ca phng php ph thuc vo kh nng chu nhit ca enzyme polymerase (do phn ng lun phi thc hin nhit khc nhau). u tin ngi ta s dng cc on Klenow ca DNA-polymerase I t E. Coli 37C (ct nh mnh DNA-polymerase I thnh on Klenow mt hot tnh exonuclease) do nhn c on cn nhn khng tinh sch v y khng phi l enzyme chu nhit. Phng php PCR chuyn sang mt bc ngot mi cng vi s pht hin enzyme Taq-polymerase c ngun gc vi khun (Thermus aquaticus) tch t sui nc nng nn chu c nhit cao. Vi enzyme ny cho php nhn c cc sn phm DNA tinh sch. iu ny cho php t ng ha tt c cc bc ca chu trnh nhn bn DNA. T my chu trnh nhit PCR ra i v c kh nng iu khin v c nhit ln thi gian. Ngy nay c rt nhiu hng trn th gii sn xut my chu trnh nhit PCR nh: Perkin Elmer, MJ, Eppendorf, ... Nhc im ca enzyme ny l khng tng hp c trnh t DNA di qu 3kb, hn na, enzyme ny khng c kh nng sa sai trong qu trnh sao chp. Hin nay c nhiu enzyme polymerase chu nhit mi c a ra th trng vi nhiu chc nng chuyn bit v hon thin hn, nh l Tthpolymerase tch t vi khun Themus thermophilus, Elogase (bao gm nhiu enzyme kt hp trong c s tham gia ca Taq-polymerese). 7.2.3.3- Mi v nhit nng chy ca mi Mi l mt ch tiu quan trng nht t c s khuch i c trng v c hiu qu cao. Chn mi phi tun theo nguyn tc sau y:

- Trnh t ca mi c chn khng c s bt cp gia mi xui v mi ngc, v cng khng to nhng cu trc kp tc. - Mi phi chn c trng cho DNA cn khuch i v khng trng vi cc trnh t lp li trn gen. - Trnh t nm gia mi xui v mi ngc khng qu ln (1kb). - di mi cn chn khong 18 n 30 nucleotide, nu mi nh hn 10 nucleotide, mi bm khng c hiu. Cn mi di hn 30 nucleotide s nh hng n c ch tng Bc 3. - Tm mi xui v mi ngc khng cch nhau qu xa, thng thng trong khong t 45C, v nhit nng chy ca mi khong 72C. 7.2.3.4- nh hng ca cc nuleotit Cn phi c bn loi nucleotide dng triphosphat nh: dATP, dTTP, dGTP, dCTP. Nng mi loi nucleotide phi dng cn bng, ng vi khong 20200M cho mi loi nucleotide. Nu mt trng thi cn bng th s gy ra li khi sao chp, nu nng cc nucleotide cao hay thp hn s dn n hin tng sao chp gi. 7.2.3.5- Mi trng phn ng Ion Mg
+2 +2
l thnh phn khng th thiu c ca phn ng PCR, nng
Mg ti u thc hin PCR t 150200M. Nng chun cho tng khon tng ng, phi c xc nh trong iu kin th nghim nht nh. Nc s dng cho phn ng PCR phi l nc tinh khit, khng cha bt k ion l no. Khng c cha cc enzyme ct hn ch v cc enzyme phn hy axit nucleic. Mi trng dung dch m phi n nh. 7.2.3.6- Thi gian v s lng chu k Qua nghin cu cho thy: tng thi gian cho mi bc ca mt chu k v ca chu k u vi cc chu k tip theo l khc nhau. Ngoi ra, thi gian cho mi phn ng cn ph thuc vo di ca on DNA cn nhn dng.

S lng chu k cho mt phn ng PCR thng thng trong khong t 30 n 40 chu k. Bi v phn ng din bin theo hai giai on: giai on u, s lng bn sao tng theo cp s nhn, v n mt gii hn no th s bn sao gim, hiu qu khuch i gim do cc nguyn nhn: - Nng nucleotide gim, - Xut hin sn phm ph, - Do cc bn sao khng bt cp vi mi m chng li bt cp vi nhau. Ngoi ra, s chu k ph thuc vo s bn mu ban u, nu s bn mu l 10 th thc hin 2530 chu k, cn s mu 10 10 th thc hin 3540 chu k. 7.2.3.7- Thit b v dng c Thit b cn p ng yu cu thay i c nhit nhanh v chnh xc. Trnh ti a s bc hi nc trong qu trnh phn ng. Tuy nhin, ng vi mi phn ng c th c cc thit b v dng c khuch i ring. 7.2.4- ng dng ca phng php PCR 7.2.4.1- u v nhc im ca phng php PCR u im: Thi gian thc hin nhanh, ch cn 3 gi l c th khuch i c mt trnh t ng quan tm. Thc hin n gin v t tn km (n c thc hin trong ng nghim plastic nh gm thnh phn ti thiu c thc hin ng thi). Yu cu v tinh sch ca mu khng cao (vt mu kh, mu vt kho c, nhng vt tch li ca ngi cht). Nhc im: Cn phi c DNA mi c trng cho DNA cn khuch i. c on mi ny t nht phi bit trc trnh t nucleotide cn khuch i. Kch thc DNA cn khuch i khng vt qu 3kb. Kh nng ngoi nhim ln (do thao tc nhiu ln). Sai st cn do s dng E-Taq-polymerase khong 10 (sai st cho mt ln sao chp). 7.2.4.2- Phm vi s dng Sn xut mu d nh du phng x (du d) vi khi lng ln, ph hp vi phng php lai gia DNA v DNA, RNA v DNA.
4 5 2 3

Khuch i s lng RNA, do lng mRNA nh di mc cho php nn ngi ta dng phng php RT-PCR tng nng mRNA n mt gii hn pht hin bng cch phin m ngc: mRNA cDNA. T cDNA, ta nh gi c mRNA. Khuch i DNA v RNA ngay ti h m: Gi l k thut insitu, k thut ny c nguyn tc nh trn, c tin hnh ngay trn lt ct m, t bo c nh trn lame trong thit b PCR c b phn to nhit cho lame. Trong nui cy m t bo, k thut PCR c dng nghin cu nhng thay i mc DNA ca cc dng m nui cy v cc cy ti sinh t m so, nghin cu s c mt ca cc gen ngoi lai trong cc cy bin np. nh lng DNA: Khi nng DNA thp, dng phng php PCR nhn ln n mc xc nh. Ngi ta xc nh c s lng bn mu ban u qua tnh ton da vo sn phm cui cng v s chu k nhn. iu quan trng l s chu k nhn khng c vt qu giai on u ca phn ng, khi m s lng bn sao cn t l thun vi s lng bn mu ban u. ng dng: Trong php y, chun on nhng bnh di truyn, nghin cu mu kho c, bo tn v duy tr nhng gen qu. 7.2.5- Nguyn l c bn ca phn ng RT-PCR (PCR ngc) Phn ng RT-PCR thc cht l phn ng nhn mt on gii hn ca khun RNA, theo nguyn l ca phn ng PCR gm c hai giai on: Giai on th nht l sao chp RNA khun thnh DNA si i nh hot ng ca enzyme sao chp ngc. Giai on ny c thc hin 5055C v thi gian l 30 pht. Giai on 2 l dng chnh DNA va sao chp lm khun cho phn ng PCR, qu trnh c thc hin theo ba bc nh trnh by trn. Sau khi phn ng kt thc, sn phm ca phn ng PCR ngc c gim xung 4C bo qun cho ti khi s dng. nh gi v pht hin sn phm PCR ngc ngi ta cng in di trn gel agarose 0,81%. V DNA chun l DNA c ct bi enzyme HindIII c di 23,1kb; 9,4kb; 6,5kb; 4,3kb, ...

7.2.6- Phng php in di DNA 7.2.6.1- Nguyn tc in di in di l k thut c s dng trong th nghim phn phn tch cc i phn t tch in. Trong phng th nghim sinh hc phn t ngi ta thng s dng phng php in di tch ly, pht hin phn t DNA nguyn vn, DNA b ct hn ch v DNA ca sn phm PCR. Axit nucleic ni chung l mt phn t tch in m, v vy, chng c th dch chuyn qua bng gel t cc m (cathode) sang cc dng (anode) di tc dng ca in trng. Trn cng mt bn gel, c cng mt dng in nhng phn t DNA khc nhau v trng lng nn khc nhau v in tch v chy c nhng qung ng khc nhau sau mt thi gian nh nhau. Sau khi phn tch bng in di, pht hin phn t DNA, ngi ta dng phng php lm hin hnh. i vi gel agarose, ngi ta nhum bng ethidium bromid. Cht ny s gn xen cc baz ca phn t DNA v pht quang di tia t ngoi. Nh vy d dng cho php pht hin v tr cc on DNA trn gel v c th phn bit c phn t DNA trn cng mt bng gel. i vi gel polyacrylamid, cc phn t thng c nh du bng ng v phng x v v tr ca chng s c pht hin bng k thut phng x t ghi. Trong in di, ngi ta s dng thang DNA chun, thng l DNA . Khi in di cho chy cng vi mu nghin cu, qua c th so snh vi DNA mu vi DNA chun bit trng lng phn t, hoc trch li c DNA khc nhau. 7.2.6.2- Cch tin hnh H thng in di bao gm ngun in, bung in di, khun gel v h thng soi chp nh. Cc bc c tin hnh nh sau: - Cn khong 1g agarose cho vo 100ml m TBE (Tris-borate-EDTA) hoc TAE (Tris-acetate-EDTA) nhit phng, khuy u v yn 1 pht, cho vo l viba (lm nng chy gel v khng to bt). - Lm ngui gel xung 5055C, thm 1l ethidium bromid, gel vo khun gel v lp lc. - Khi gel ngui, t khun gel vo bung in di, tho lc v m chy vo khay gel sao cho m cao hn mt gel khong 25 mm.

- Np DNA mu v DNA chun vo cc ging, y np v cm in cc. - in di trong khong 30 pht vi in th 100120V. - Chp hnh nh gel di nh sng UV xem kt qu. Lu : Ty thuc vo kch thc ca DNA mu m ngi ta chn loi gel (agarose, polyacrlamid), nng gel, loi m (TBE hoc TAE). m TAE cho phn gii cao cc phn on ln hn 4 kb, trong khi m TBE c phn gii cao t 0,1 n 3kb. Ngoi ra, phn gii cc phn on DNA cn ph thuc vo phng php in di, ti u in th, ti u lng DNA np v m np. Thuc nhum ethidium bromid (EtBr) l mt thuc nhum hunh quang m c th nhn bit c hai loi DNA si n v si kp. Mc d vy, i lc lin kt vi DNA si n tng i thp hn so vi DNA si kp. DNA nhum EtBr c nhn bit bng tia cc tm. Ti bc sng 254nm, nh sng UV c hp th bi DNA v chuyn sang thuc nhum. Ti bc sng 302nm v 366nm, nh sng UV c hp th bi chnh thuc nhum. Trong c hai trng hp, nng lng c pht x ng vi tia ti bc sng 590nm trong vng vng ca quang ph thy c. EtBr l cht gy t bin mnh, cn phi mang gng tay khi thao tc vi dung dch thuc nhum ny v cc gel nhum. to phn gii cao r nt, cc vch v nn nht nn nhum gel vi EtBr ngay sau khi in di. 7.3- CC PHNG PHP LAI PHN T 7.3.1- Phng php Southern blot Phng php Southern blot c E. Southern pht minh vo nm 1975, cho php nghin cu DNA ca b gen, kim tra kt qu chuyn gen hoc kim tra s c mt ca mt gen no trong b gen ca t bo. thc hin c qu trnh lai, u tin, ngi ta phi tch DNA b gen ca t bo. S dng enzyme ct gii hn ct phn t DNA thnh nhng on nh, in di trn gel agarose tch nhng on c kch thc khc nhau. Gy bin tnh DNA trn gel bng dung dch NaOH-0,5M, sau , chuyn DNA t gel sang mng lai (nitrocellulose). Trong qu trnh chuyn, v tr ca DNA phi c gi nguyn.

DNA c nh trn mng c lai vi mu d c nh du phng x, nhit 65C v thi gian lai khong 38 gi. Sau qu trnh lai ngi ta ra mng lai loi b nhng mu d khng bt cp chuyn bit vi DNA c nh trn mng lai.
8 7 6 5 4 3 2 1 1: Dung dch m 20X.SSC 2: Ga 3: Bc giy lc 4: Gel agarose 5: Mng lai 6: Giy thm 7: Tm knh 8: Vt nng
Hnh 7-4: K thut chuyn DNA t gel agarose ln mng lai Cui cng, ngi ta dng k thut phng x t ghi nh v cc phn t lai. Trong k thut ny, ngi ta t mt phim nhy cm vi tia x p st vo mng lai. Cc phn t lai c nh du phng x s tc ng ln phim v kt qu c th hin qua cc chm en trn phim (Hnh 7-4). Cc ng dng quan trng ca Sourthern blot: - Lp bn gii hn ca mt gen. - Pht hin cc a dng trnh t ca cng mt gen cc chng hay cc c th khc nhau qua s so snh bn gii hn ca chng. - Pht hin cc t bin mt on, t bin im hay ti t hp trn mt gen v chng lm thay i bn gii hn. 7.3.2- Phng php Northern blot Phng php lai Northern blot l phng php lai RNA ca t bo vi mu d DNA. Phng php ny c s dng xc nh kch thc v hm lng ca mt mRNA c trng trong mt hn hp RNA. V nguyn tc ging nh lai Southern blot, c tin hnh nh sau:

RNA ( c lm bin tnh) s c phn tch theo kch thc nh in di trn gel agarose c cha cht lm bin tnh. Cc cht lm bin tnh c tc dng ngn cn s hnh thnh cu trc bc hai ca RNA sau khi bin tnh. V do , khng cn tr s di chuyn cng nh s tch ca cc RNA trn gel. Sau , RNA c chuyn ln mng lai. Nhng RNA c nh trn mng lai c em lai vi mu d DNA c nh du phng x to phn t lai RNA-DNA. Cc phn t lai c pht hin nh k thut phng x t ghi. 7.3.3- Lai ti ch Lai ti ch l phng php lai phn t, trong , trnh t axit nucleic cn tm nm ngay trong t bo hay trong m. Lai ti ch cho php nghin cu axit nucleic m khng cn qua giai on tch chit chng ra khi m, t bo. Cc ng dng ca kiu lai ny rt a dng i t k thut nh v gen trn nhim sc th, pht hin dng vi khun ti t hp trong phng php to dng n vic nghin cu mt mRNA chuyn bit trong t bo v m. Phng php ny t tn km hn l lai Southern blot, mu d c nh du bng hunh quang thay cho mu d nh du phng x. Ty tng loi t bo v m c th thc hin cc phng php lai ti ch khc nhau. 7.3.3.1- Lai trn khun lc Phng php ny c s dng pht hin dng vi khun c mang vector ti t hp cn tm trong mt ngn hng gen. Ngn hng gen c tri trn mt thch ca hp petri. Sau khi cc khun lc t kch thc nht nh, ngi ta thu ly du n ca chng bng cch p mt mng lai trn mt thch. Mi khun lc s li vi t bo vi khun trn mng lai. Sau x l mng lai bng NaOH lm v t bo vi khun v lm bin tnh DNA. Kt qu phng x t ghi ca du n cho php xc nh dng vi khun cn tm trn hp pectri ban u. Dng ny s c thu nhn v phn tch. 7.3.3.2- Lai trn nhim sc th Phng php ny cung cp thng tin chnh xc v v tr v s phn b ca mt trnh t DNA cn tm trn nhim sc th nh mt mu d chuyn bit. Chn t bo k gia ca qu trnh phn bo khi cc nhim sc th c kch thc ln nht (cc nhim sc th ny thng c ngun gc t bch cu). Bng cc k thut x l c nh t bo trn lam knh, nh v hnh dng v v tr ca nhim sc th. S dng enzyme RNase v enzyme protease K loi

b RNA v protein. Nhim sc th c nh trn lam c em lai vi mu d DNA hoc RNA c nh du phng x. S dng k thut phng x t ghi pht hin phn t lai (phn t lai trn lam c ph mt huyn dch nhy cm vi tia x). Sau mt thi gian, cho tia x tc ng ln huyn dch, lam c em quan st di knh hin vi. Ti cc im c cc phn t lai xut hin cc chm en trn lp huyn dch. 7.3.3.3- Lai trn t bo v m Lai trn t bo v m thng c s dng pht hin cc mRNA. Cc loi mRNA hot ng cc giai on khc nhau trong qu trnh pht trin t bo v c th. Lai trn t bo v m nhm nghin cu giai on hot ng ca mRNA, t , tm hiu hot ng ca mt gen, mi lin quan gia phin m v gii m, nh v cc gen cn nghin cu trn nhim sc th. M hoc t bo c x l bng phng php m, t bo hc nh: c nh, kh nc, tm paraffin, ct thnh nhng lt mng (710m) tri trn lam. X l vi enzyme protease loi b protein, sau x l vi enzyme DNase loi b DNA. Lai vi mu d c nh du phng x hoc hunh quang. Sau ph mt lp huyn dch nhy vi phng x hoc hunh quang mt thi gian thch hp cho phn ng xy ra, quan st kt qu lai di knh hin vi. Trong t bo nhng v tr c phn t lai c pht hin bng cc chm en. Phm vi s dng ca phng php: - i tng nghin cu c t chc phc tp bao gm nhiu tp hp cc t bo khc nhau nh b no. - Cc phng php min dch t bo cho php pht hin mt protein chuyn bit thng qua khng th c trng ca n. Khi phi hp nhng phng php min dch t bo v lai ti ch, ngi ta s pht hin ng thi mRNA v protein ca n, t , xc nh c mi tng quan gia hot ng phin m v dch m ca mt gen. - Bng cch lai nhiu lt ct lin tc (c cu trc gn nh ng nht) vi nhiu mu d khc nhau, ngi ta c th xc nh v tr, s phn b v tng tc gia cc mRNA cng tham gia vo mt qu trnh sng.

7.4- PHNG PHP GII TRNH T CA AXIT NUCLEIC Khi nghin cu v gen, vic xc nh c v tr ca gen trn nhim sc th cha cho php kt lun v bn cht ca n nh gen tng ng vi gen g, chc nng iu ha hay m ha cho protein no. Nh vic gii trnh t gen m ngi ta gii quyt c nhng kh khn trn. C hai phng php gii trnh gen l phng php ha hc ca Maxam-Gilbert v phng php enzyme ca Sanger v cng s. 7.4.1- Phng php ha hc ca Maxam-Gilbert Vo nm 1977, Maxam v Gilbert ln u tin pht minh ra phng php gii trnh t gen bng phng php ha hc. Nguyn tc ca phng php l da trn phn ng ha hc thy gii c hiu, cc DNA khng t xon li vi nhau, to thnh tp hp nhiu phn on c kch thc khc nhau. Trc ht, phn t DNA c nh du ng v phng x P u 5 ca mch n, to nhng on nh du c th pht hin bng hnh phng x. X l ha hc c hiu phn hy c trng mt loi nucleotide ca mch DNA nh du phng x, to cc on oligonucleotide c chiu di hn km nhau 1 nucleotide c pht hin bng in di. Kt qa cc phn ng ha hc x l mch DNA c pht hin bng in di trn gel polyacriamid c th xc nh c trnh t mch n. Mch n nh du phng x c th x l theo 4 nhm phn ng: - Nhm phn ng th nht x l mch n DNA bng dimethyl sulphat lm t mch ti G. - Nhm phn ng th hai x l mch n DNA bng axit (pH=2) gy t mch n ti A hoc G. - Nhm phn ng th ba x l mch n DNA bng hydrazin gy t mch n ti T v C. - Nhm phn ng th 4 x l mch n DNA bng hydrazin vi nng mui cao lm t mch n ti C.
32

Kt qu: Cc phn ng to thnh cc on DNA ct ngu nhin, c kch thc khc nhau. in di trn gel polyacriamid (Hnh 7-5), c kt qu in di bng my phng x t ghi ta thu c trnh t nucleotide ca mch n DNA.
G G+A T+C C
ACACTG
ACACT
ACAC
ACA
AC
A
+
Hnh 7-5: S gii trnh gel theo phng php ho hc V d, on mch n DNA nh du phng x P , u 5` nucleotide A c trnh t : 5`P ACACTG Sau khi x l bng phng php ha hc v ct mch n ni trn ta thu c cc phn on sau: - Kt qu nhm 1 ct ti G: P ACACT P ACACTG - Kt qu phn ng nhm 2 ct ti A hoc G: P AC P ACACTG P ACACT P ACACTG - Kt qu phn ng nhm 3 ct ti C hoc T: P A P ACA P ACACTG P ACAC P ACACTG
32 32 32 32 32 32 32 32 32 32 32 32 32

- Kt qu phn ng nhm 4 ct ti C: P A P ACA P ACACTG Tng hp cc kt qu, ta thu c trnh t cc nucleotide trn mch n DNA, t ta bit c trnh t sp xp cc nucleotide ca gen. 7.4.2- Phng php enzyme s dng cc dideoxynucleotide Phng php ny c F. Sanger v cng s pht minh vo nm 1977. Nguyn tc ca phng php dideoxy l da vo s tng hp mch b sung cho trnh t cn xc nh nh hot ng ca enzyme DNA-polymerase. Vi vic s dng thm cc dideoxynucleotide (ddNTP) vi cc deoxynucleotide (dNTP) thng thng, kt qu tng hp cng l s hnh thnh mt tp hp nhiu on DNA c kch thc khc nhau. Trong phn ng s dng on DNA mi mch n khong 20 nucleotide, bn loi dNTP (trong c mt loi nh du bng ng v phng x) v b xung thm 1% ddATP (hoc ddCTP, ddGTP, ddTTP) cho mi loi phn ng. Cc ddNTP mt hai nguyn t oxy C3 v C2 khi mch n DNA ang tng hp c gn 1 ddNTP phn ng ngng tng hp. Mi loi phn ng c thc hin ring r, c DNA khun, DNA mi, y cc loi dNTP, enzyme Taq DNApolymerase, dung dch m v c thm khong 1% mt loi ddNTP. Kt qu phn ng tng hp nn cc on oligonucleotide di ngn khc nhau mt nucleotide v c nhn bit nh phng php in di (Hnh 7-6). Tng hp kt qu phn ng 4 ng, thu c trnh t cc nucleotide ca mch n, c th bit c trnh t sp xp cc nucleotide trong gen.
32 32 32
TGAGGTC
TGAGGT
TGAGG
TGAG
TGA
TG
Hnh 7-6: S gii trnh gen theo phng php Sanger
V d: Gii trnh on gen: - Mch cn tng hp l - Kt qu phn ng theo loi A:
5`- AGTCCAG-3` 3`- TCAGGTC-5` TCAdd TCAGGTC TCAGdd TCAGGdd TCAGGTC TCdd TCAGGTCdd TCAGGTC
- Kt qu phn ng loi G:
- Kt qu phn ng loi C:
- Kt qu phn ng loi T:
Tdd TCAGGTdd TCAGGTC
7.4.3- Phng php xc nh trnh t nucleotide bng my t ng Trong k thut ny, ngi ta khng nh du bng cc ng v phng x m bng ha cht (cc fluochrome). Mi loi ddNTP c nh du bng mt fluochrome c mu khc nhau. Nh vy, tt c cc oligonucleotide cng chm dt ti mt loi ddNTP s c cng mt mu. Sau khi in di trn gel polyacriamid, kt qu s c x l qua mt h thng vi tnh.
TI LIU THAM KHO

1- i Duy Ban (19980, Cng ngh ADN trong iu tr gen cc bnh him ngho, Nxb Y hc, H Ni. 2- Bi Ch Bu, Nguyn Th Lang (1999), Di truyn phn t - Nhng nguyn tc c bn trong chn ging cy trng, Nxb Nng nghip, H ni. 3- Nguyn Hu Chn ch bin (1999), Nhng vn ho sinh hc hin i, Nxb KH&KT, H Ni. 4- Phm Th Trn Chu, Trn Th ng (1997), Ho sinh hc, Nxb Gio Dc, H Ni. 5- Nguyn Ln Dng ch bin, Nguyn nh Quyn, Phm Vn Ty (1997), Vi sinh vt hc, Nxb Gio dc, H Ni. 6- H Hunh Thu Dng (1998), Sinh hc phn t, Nxb Gio dc, H ni. 7- Nguyn Nh Hin (2002), Di truyn v cng ngh t bo soma, Nxb KH&KT, H Ni. 8- nh Ho ch bin (1996), Gio trnh ho sinh, i hc Y Dc, TP HCM. 9- Phm Thanh H (2000), Di truyn hc, Nxb Gio dc, H Ni. 10- Nguyn nh Huyn (1998), Sinh hc phn t ADN, Nxb KH&KT, H Ni. 11- V Th Thng Lan (2002), Sinh hc phn t, Nxb i hc Quc gia, H Ni. 12- Nguyn Th Lang (2002), Phng php c bn trong nghin cu cng ngh sinh hc, Nxb Nng nghip-Chi nhnh TP H Ch Minh. 13- L nh Lng, Phan C Nhn (1993), Di truyn hc, Nxb KH&KT, H Ni. 14- Phan C Nhn (1992), Mt s vn v di truyn hc (hin i), Nxb Gio dc v o to, H Ni. 15- Phan C Nhn (1998), Sinh hc i cng, Nxb i hc Quc gia, H ni.

16- Khut Hu Thanh (2003), C s di truyn hc phn t v k thut gen, Nxb KH&KT, H Ni. 17- Nguyn c Thnh (2000), Nui cy m t bo thc vt - Nghin cu v ng dng, Nxb Nng nghip, H Ni. 18- L Ngc T (1997), Ho sinh cng nghip, Nxb KH&KT, H Ni. 19- L Ngc T, Ngc Lin, ng Th Thu (2002), T bo v cc qu trnh sinh hc, Nxb KH&KT, H ni. 20- Nguyn Vn Uyn ch bin, Nguyn Tin Thng (2000), Nhng kin thc c bn v cng ngh sinh hc, Nxb Gio dc, H Ni. 21- C.Vili v Dethio (1979), Cc nguyn l v cc qu trnh sinh hc, Nxb KH&KT, H Ni. 22- Emil L. Smith (1983), Principles of Biochemistry, Mc. Graw-Hill Book Company (7th Edition), New Dehi. 23- Emil L. Smith, Robert L. Hill (1983), Principles of biochemistry -General aspects, McGraw-Hill Book Company, New Dehi. 24- Hartl Daniel L., Freifelder David, Snyder Leon A. (1988), Basic genetics, Johnes and Bartlett Publishers Inc., USA. 25- Rastogi S. C. (1996), Biochemistry, Tata McGraw-Hill Publishing Co.Ltd, New Dehi. 26- Ren Scriban coordonnateur (1982), Biotechnologie, Technique et documentation lavoisier, Paris. 27- The national medical series for indepenent study (1996), Genetics, William & Wilkins, USA. 28- Watson James D., Hopkins Nancy H., Roberts Jeffrey W., Steiz Joan Argetsinger, Weiner Alan M. (1987), Molecular biology of the gene, The Benjamin/Cummings Publishing Co. Inc., USA. 29- William M. Fogarty (1983), Microbial enzymes and biotechnology, Applied Science Publishes Ltd, London & New York.

MC LC
Phn I- C s di truyn M u- Lc s pht trin ca di truyn hc v k thut di truyn 1.1- Lnh vc nghin cu ca di truyn hc v cng ngh di truyn 1.2- Giai on di truyn sau Mendel 1.3- S ra i ca cng ngh di truyn 1.4- ngha khoa hc v thc tin ca cng ngh di truyn 1.4.1- Trong y dc 1.4.2- Trong nng nghip 1.4.3- Trong cng ngh thc phm Chng I - Bn cht ca vt cht di truyn 1.1- Axit nucleic - Vt cht di truyn ca mi sinh vt 1.1.1- Thnh phn cu to ho hc ca axit nucleic 1.1.2- Cu trc phn t DNA 1.1.3- Tnh cht ca DNA 1.1.4- Cc th nghim chng minh DNA l vt cht di truyn 1.2- Cc RNA 1.2.1- Cu to ha hc v c im chung ca RNA 1.2.2- Cc loi RNA 1.3- M di truyn 1.3.1- Khi nim v m di truyn 1.3.2- c tnh ca m di truyn 1.4- Vt cht di truyn ca mt s nhm sinh vt 1.4.1- Vt cht di truyn ca virus 1.4.2- Vt cht di truyn ca vi khun 1.4.3- Vt cht di truyn ca eucaryote Chng II - Hot ng v s biu hin ca gen 2.1- Gen l n v chc nng ca b my di truyn 2.1.1- Cc quan nim v gen 2.1.2- Cu trc ca gen 2.2- S sao m 2.2.1- L thuyt v sao m DNA theo khun 2.2.2- Qu trnh sao m 2.2.3- Sao m DNA si kp dng vng ca t bo procaryote 2.2.4- Sao chp m t bo eucaryote 2.2.5- Sa sai DNA trong t bo 2.3- S phin m 2.3.1- c im chung ca qu trnh phin m 2.3.2- Phin m tng hp mRNA procaryote 2.3.3- Phin m tng hp mRNA eucaryote 2.3.4- Phin m tng hp tRNA 1 2 3 4 5 6 7 9 14 20 21 25 26 31 32 33 34 38 38 39 41 44 48 50 53 55 56 61 65

2.3.5- Phin m tng hp rRNA 2.4- Dch m 2.4.1- c im chung ca qu trnh dch m 2.4.2- S hot ho v vn chuyn axit amin 2.4.3- Hng c m v hng ko di si polypeptide 2.4.4- Cc giai on ca qu trnh dch m 2.4.5- Polyribosome 2.5- iu ho biu hin gen (iu ho sinh tng hp protein) 2.5.1- Mc ch ca iu ho biu hin gen 2.5.2- Cc yu t iu ho biu hin gen 2.5.3- M hnh iu ho biu hin gen vi khun 2.5.4- M hnh iu ho biu hin gen eucaryote 2.5.5- S bit ha t bo Chng III - Bin i vt cht di truyn 3.1- Khi nim v bin d 3.2- t bin 3.2.1- Tc nhn gy t bin 3.2.2- t bin gen 3.2.3- t bin nhim sc th 3.3- Ti t hp Phn II- Nhng nguyn l c bn ca cng ngh di truyn Chng IV- Cc enzyme s dng trong cng ngh di truyn 4.1- Cc enzyme gii hn 4.1.1- Hin tng gii hn v h thng hn ch - ci bin 4.1.2- Cch gi tn ca enzyme gii hn 4.1.3- Cc loi enzyme gii hn 4.1.4 - Cc loi RE trong nhm II 4.1.5- ng dng ca RE trong cng ngh di truyn 4.2- Cc loi nuclease 4.2.1- Phn loi 4.2.2- Mt s nuclease thng dng v ng dng ca n 4.3- Enzyme kt ni 4.3.1- Ligase 4.3.2- Cc enzyme dephosphoryl ho 4.3.3- Cc enzyme phosphoryl ho 4.4- Cc enzyme tng hp 4.4.1- Enzyme sao chp DNA 4.4.2- Enzyme phin m ngc 4.4.3- Cc enzyme tng hp RNA (RNA-polymerase) 4.4.4- Enzyme terminal-transferase Chng V - Vector chuyn gen 5.1- Khi nim v vector 5.1.1- S chuyn gen 66 67 68 70 74 75 76 80 82 84 86 88 92 94 96 98 99 100 104 105 106 108 109 110 111 112 113 -

5.1.2- Vector 5.2- Nhng yu cu ti thiu ca mt vector chuyn gen 5.3- Mt s ng dng ca vector chuyn gen 5.4- Cc vt ch thu nhn cc vector chuyn gen 5.5- Cc loi vector chuyn gen 5.5.1- Vector chuyn gen l plasmid 5.5.2- Cc vector chuyn gen l phage 5.5.3- Cc vector chuyn gen khc 5.5.4- Cc vector l virus ca eukaryote Chng VI - Cng ngh DNA ti t hp v s tch dng 6.1- Cng ngh DNA ti t hp 6.1.1- DNA ti t hp 6.1.2- Cc cng on chnh to DNA ti t hp 6.1.3- Cc phng php to DNA ti t hp 6.2- Mt s phng php a DNA ti t hp vo t bo ch 6.2.1- Ha bin np 6.2.2- in bin np 6.2.3- Bin np t bo trn 6.2.4- Phng php bn gen 6.2.5- Phng php vi tim 6.2.6- Ti np 6.3- K thut tch dng (to dng) 6.3.1- Mc ch ca s tch dng 6.3.2- Cc bc c bn ca phng php tch dng 6.3.3- Mt s phng php xc nh dng cn tm 6.4- Ngn hng gen 6.5- Ngn hng cDNA 6.5.1- Thit k ngn hng cDNA 6.5.2- Sng lc ngn hng cDNA Chng VII - Mt s phng php nghin cu trong cng ngh di truyn 7.1- Cc phng php tch chit axit nucleic 7.1.1- Phng php tch chit DNA vi khun 7.1.2- Phng php tch DNA plasmid 7.1.3- Tch DNA ca t bo khc 7.1.4- Phng php tch ARN tng s v mRNA 7.1.5- Tch v thu nhn gen 7.2- Phng php nhn bn (PCR) 7.2.1- Nguyn tc ca phng php PCR 7.2.2- Cc bc tin hnh th nghim 7.2.3- Cc yu t nh hng n phn ng PCR 7.2.4- ng dng ca phng php PCR 7.2.5- Nguyn l c bn ca phn ng RT-PCR (PCR ngc) 7.2.6- Phng php in di DNA 114 115 116 121 125 128 129 132 135 140 141 142 143 145 148 149 150 151 152 153 154 156 157 158 161 163 164 165

7.3- Cc phng php lai phn t 7.3.1- Phng php Southern blot 7.3.2- Phng php Northern blot 7.3.3- Lai ti ch 7.4- Phng php gii trnh t ca axit nucleic 7.4.1- Phng php ha hc ca Maxam-Gilbert 7.4.2- Phng php enzyme s dng cc dideoxynucleotide 7.4.3- Phng php xc nh trnh t nucleotide bng my t ng Ti liu tham kho Mc lc 166 167 168 170 172 173 174 176

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Tai lieu chia se tai: wWw.SinhHoc.edu.

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BN CHT CA VT CHT DI TRUYN

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Tai lieu chia se tai: wWw.SinhHoc.edu.vn Axit nucleic (DNA v RNA)

ng pentose - Deoxyribose - Ribose

Baz nit - Purine - Pyrimidine

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Adenine (A) (6-aminopurine)

Guanine (G) (2-amino 6-oxypurine)

Cytozine (C) (2-oxy-4-aminopyrimidine)

Thymine (T) (2,4 dioxy-5 methylpyrimidine)

Uracil (U) (2,4 dioxypyrimidine)

Hnh 1-2: Cng thc cu to ca cc baz nit

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Adenosine 5-monophosphat (Adenosine monophosphat) (AMP)

Uridine 5-monophosphat (Uridine monophosphat) (UMP)

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Dng imin t gp (2 dng quay)

Dng enol t gp (2 dng quay)

Hnh 1-5: V tr c th hnh thnh lin kt hydro ca cc dng baz nit

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Hnh 1-6: Cu to ca 2 si polynucleotide v s bt cp b sung ca cc baz nit

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3' 3' 3' 3' 3'

Hnh 1-7: M hnh cu trc phn t DNA

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Dng R v dng S T bo vi khun dng R

Ch c dng R T bo vi khun dng R

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