Tch dng gen t DNA

I . Khi nim T{ch dng gen l| KN ch mt nhm c{c phng ph{p c SD : Ph}n lp trnh t DNA c hiu t hn hp c{c PT DNA ban u v| sao chp DNA ln s lng ln tin h|nh ph}n tch v cu trc v| CN ca gen tng ng K thut t{ch dng bao gm vic phi c|i mt mnh (chui) DNA l v|o mt vector bng phng ph{p ha sinh. Sau , a ph}n t lai n|y v|o t b|o ch la chn bng phng ph{p bin np hoc ti np.

II-Nguyn liu

1- DNA l cn tch dng

2 - Enzym

3 - Vector chuyn gen

4 - H thng t bo ch

1- DNA cn tch dng

L on DNA l, c thu nhn t TB sinh vt, c chit tch, lm sch v kim tra theo phng php chit tch DNA

2 - Enzym5 nhm enzym:1. Cc enzym lm t gy lin kt photphodieste (enzym gii hn, enzym Mungbean nuclease) 2. Cc enzym ni khung phn t DNA(E. coli ligase, T4 DNA ligase, T4 RNA ligase) 3. Cc enzym b sung hoc loi nhm phosphate 4. Cc enzym tng hp mi lin kt phosphodieste mi trong phn t DNA (DNA polymerase, Reverse transcriptase) 5. Cc enzyme tham gia bo v, phn gii, xon v gin xon DNA (methylase)

2.1- Enzym ct gii hn (RE) Enzym gii hn l enzym c kh nng nhn bit nhng on trnh t ADN nht nh v ct ADN ngay im ny hay im k cn. VT: ct DNA v tr c trng RE ct lin kt phosphodieste lm cho phn t ADN b t th|nh nhiu on nh

ADN ca k ch khng b ct bi RE nh c{c v tr ct c methyl ha (enzyme methylase gn nhm CH3 vo base A hoc C ti v tr ct)

C hai kiu ct ca enzym gii hn l kiu ct to u bng (blunt ends) v u sole (u dnh - cobesive ends).

Enzym gii hn ct u bng khng t ni cc on ADN li vi nhau( sd enzym ni ligase) Enzym gii hn ct u sole to u dnh. Sau khi ct chng c th t ni li vi nhau theo nguyn tc b sung

2.2- Cc loi enzym khcENZYMDNA polymerase Enzym phin m ngc Enzym ni ligase Enzym methylase

VAI TRNi cc nu vi nhau theo NTBS -> mch DNA TH mch DNA b sung vi mach khun RNA theo chiu 5-3 Ni cc on acid nucleic vi nhau bng lk phosphodieste Thay i tnh c hiu ca mt s enzym gii hn

3- Vector tch dng Vector l mt phn t DNA thng c dng vng, mang nhiu c tnh trong c kh nng xm nhp vo t bo vi khun v t sao chp.

3.1- Plasmid u im Cu trc n gin, kch thc nh D tinh sch, d phn tch on ti t hp C th nhn ln vi s lng ln, tc nhanh Nhc im i khi hiu sut bin np vo TBVC thp Khng hiu qu khi bin np Eukaryote Khng tch dng c cc phn on DNA c kch thc ln (>10kb)- Plasmid l nhng on DNA ngn (2-5kb), dng vng, nm ngoi nhim sc th, c vi khun.

3.2- Thc khun th (Phage) u im: Kh nng xm nhp t bo ch nhanh. C th tch dng c prokaryote v eukaryote. C th mang cc on ci n ~ 20 kb. D bo qun do cc ht virut bn nhit lnh (vd. 4 o C). Nhc im: Kch thc ln hn plasmid, nn phn tch phc tp hn. S bn sao phage hnh thnh trong mi t bo thp hn s bn sao ca plasmid. Phage l| thut ng ch c{c loi virut k sinh trn vi khun. C kh nng mang gen t t bo vi khun cho sang t bo ch nhn

3.3- NST nh}n to ca nm men (YAC) C im khi u sao chp (origin of replication) ca nm men, trnh t tm ng, v u tn cng (telomeres) ging nh cu trc mt NST ca TB chn hch

C th chuyn cc on ADN kch thc 1501000kb

4- H thng TB vt ch Mc ch: to DNA ti t hp

C th s dng c TB nhn s v nhn chun lm TBVC Yc: D nui cy gi v nhn ging, chp nhn c nhiu loi vector.

VK E.coli

Nm men

1- Tch lp cc DNA cn to dng 2- Chn vector

III- Quy trnh

3- To DNA ti t hp (Vector ti t hp) 4- Chuyn DNA ti t hp vo t bo ch 5-Cc phng php xc nh dng cn tm

1- Tch lp c{c DNA cn to dng

Chn v ct DNA ca t bo cho bng mt enzyme ct hn ch (RE). Khi s dng enzym ct (RE), c th to ra u dnh hoc u bng. u dnh thng c dng hn do tnh chnh xc.

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

2- Chn vector Chn vector da v|o kch thc v| bn cht on ADN Yu cu ca vector tch dng: Kch thc tng i nh so vi NST ca TB ch C th nh}n i c lp trong TB ch C vng nhn bit enzym gii hn C th nhn bit bng gen ch th hoc gen {nh du. Trnh t nu x{c nh Phi thch hp vi t b|o vt ch C v tr gn ph}n t DNA ngoi lai (polycloning site)

La chn vector tch dngLoi vector tch dngPlasmid Phage Phagemid Cosmid BAC (thit k t mt phn DNA E.coliv cc plasmid) TAC (thit k t mt phn on vir gen ca Ti plasmid) BiBac (thit k t mt phn DNA b gen ca Agrobacterium) PAC (b gen ca phage P1+plasmid) YAC (thit k t mt phn DNA b gen ca nm men)

Kch thc on gen ch c th ci1-5 kb 15-25 kb 20-10kb 30-50 kb 100-300 kb 100-300 kb 100-500 kb 100-300 kb 2000

3- To DNA t{i t hp (Vector t{i 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 (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.

Phng ph{p n gin dng u lch

4- Chuyn DNA t{i t hp v|o t b|o chMc ch : s dng b m{y ca t b|o ch sao chp vector t{i t hp th|nh 1 s lng ln bn sao. C 2 phng ph{p chuyn DNA t{i t hp v|o TB ch: - Bin np - Ti np

Cc phng php chuyn DNA4.1- Bin np L hin tng chuyn vt cht di truyn trc tip t t bo th cho sang t bo th nhn vi vector chuyn gen l plasmid Bin np l qu trnh 1 c th ch c th tip nhn 1 phn t DNA ngoi lai t mi trng bn ngoi

Cc phng php chuyn DNA4.1- Bin np C nhiu phng php bin np: s dng sng bn gen, bin np TB trn, vi tim, xung in (nm men), sc nhit (vi khun), Vi khun c kh nng bin np t nhin gi l cc vi khun kh bin di truyn (E.coli c th kh bin khi c x l vi ion Ca2+). Sau khi bin np , thng ch c 1 phn nh t bo ch c vector ti t hp.

Cc phng php chuyn DNA4.1- Ti np* L hin tng chuyn vt cht di truyn trc tip t t bo th cho sang t bo th nhn * c thc hin vi vector chuyn gen c ngun gc l vius nh phage, comid.thc khun th )

5- Cc phng php xc nh dng cn tm5.1- Phng php lai axit nucleic

5.2- Phng php s dng khng th

5.3- Phng php pht hin do thay i kiu hnh

5.1- Phng php lai axit nucleicNguyn tc Da vo kh nng bin tnh khi nhit tng v hi tnh khi h nhit t t. Khi tng nhit ln qu nhit sinh l ( 8095oC), 2 si n DNA s tch di nhau do lin kt hidro 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 xy ra khi 2 trnh t DNA hon ton b sung cho nhau

5.2- Phng php s dng khng thNguyn tc da vo phn ng c trng ca 1 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 Cch tin hnh Chn khng th c trng cho protein Tch protein c biu hin trong qu trnh to dng protein vi khng th c trng Tin hnh nhn bit thng qua cc du hiu ca phn ng c trng gia protein v khng th

5.3- Phng php pht hin do thay i kiu hnhNguyn tc Da vo s thay i mu sc ca khun lc, chng t rng khun lc cha gen l Cch tin hnh Chun b 2 mu th nghim m plasmit to dng c cha gen lacZ , 1 mu so snh cn mu kia ci DNA l vo gen lacZ Nui cy c 2 mu trong mi trng c cht cm ng X-Gal (5brom-4chloro-3indolyl D- galactopynoside) cht cm ng ny b phn hy bi enzim -galactosidase to ra galactose v X ( dn xut indol) dn xut ny cho mu xanh m Qua quan st t thy , plasmit khng cha gen l cho khun lc mu xanh, cn plasmit th 2 cho khun lc mu trng -> chng t n cha DNA l c ci trong gen lacZ v chn nhng khun lc mu trng

IV-Mc ch ca s t{ch dng Thit lp ng}n h|ng b gen. Thit lp ng}n h|ng cDNA. Sn xut protein, enzyme. Sn xut vaccine. Sn xut kh{ng sinh.

1- Ngn hng gen (genomic library)- L tp hp cc trnh t DNA ti t hp c cc chui m ha ln cha m ha , bao trm cc h gen ca sinh vt ang nghin cu. - ngha : Ngn hng h gen c ngha quan trng trong vic lp bn di truyn, gii m trnh t h gen.

2- Ngn hng cDNA (cDNA library)-L tp hp cc bn sao cua cDNA t mDNA -Mang tnh t bo cao -Mc ch: Nghin cu s biu hin ca mt gen xc nh cng nhng vn lin quan iu ha gen. Nghin s tng tc gia cc gen trong mt qu trnh sng