1. 2. 3. 4. 5.

K thut gen l g ? Nu cc lnh vc ng dng ca k thut gen? Gen l g? Chc nng v c im cu trc b gen? Nu cc phng php c bn trong k thut gen? So snh cc phng php? Trnh by tch dng v biu hin gen? Trnh by cc cng ngh c bn trong k thut gen v ng dng? _____________________________

K thut gen gm thao tc trn vt cht di truyn, ci to cu trc gen hoc b gen, to gen or protein mi, phc v li ch chnh ng v nhn o. Cc lnh vc ng dng ca k thut gen: 1. Nghin cu c bn :chc nng gen v protein, c ch iu ha hot ng gen, kin to gen, nhn bn v bo tn, proteomic, genomic chc nng gen v di truyn. 2. Trong y hc: sn xut insulin, vaccin, hormon, khng sinh,... + liu php gen phng v iu tr bnh a. Sn xut: Insulin: NST 11p, l protein nh 51 aa, gm hai chui peptidA(21) v B(30) ni vi nhau bng cu S, vai tr trong chuyn ha G thnh glycogen, thiu gy tiu ng , kh gic mc, l lot do ri lon chuyn ha. Sn xut : 1. Tch DNA t t bo tuyn ty 2. Tch dng on gen m ha chui A v on m ha B 3. Bin np vo E.coli nui cy to sinh khi 4. Tch chit cc phn t protein 5. Ct v tinh sch pr thu A v B 6. Phi trn to cu S 7. Hon thin sn phm. Phng php miniproinsulin MPI: 1. Tch chit mRNA phin m ngc to cDNA kp, thay on gen to gen m ha proinsulin. 2. Gn on gen m ha miniinlulin vo vector biu hin. Tng hp trong E.coli. 3. To dng E.coli mang vector ti t hp 4. Nui cy thu miniinsulin gp cun ng 5. Thu nhn v tinh sch, ct b on tha thu insulin c hot tnh. 6. Th hot tnh v hon thin Sn xut hormon: 1.Tch hGH-mRNA phin m ngc to cDNA 2.Tch dng hGH-cDNA vi vector thch hp3.Biu hin gen trong t bo vi khun E.coli4. Tch chit v tinh sch hGH v th nghim trn ng vt Vaccin: Vaccin truyn thng:Ag b bt hot hoc gim c lc: vi khun, virus Vaccin hin i: mnh Ag, vaccine ti t hp: tng hp v virus, vaccin DNA Tch dng gen m ha v protein cn To vector biu hin Chuyn vo t bo ch Ln men to sinh khi Tch thu pr cn, tinh sch Hon thin Liu php gen phng v tr bnh: Cc k thut c bn ca liu php gen s dng: chuyn gen trong v ngoi c th sng: cc k thut t bin v chuyn gen Cc k thut chun on bnh trc khi sinh, cc chip sinh hc pht hin mu bnh phm, cc k thut chun on t bin gen, chun on DNA. Chun on trc sinh cc bnh: thiu mu hng cu lim, thiu mu, bnh di truyn, bnh thiu ht min dch Pht hin mu bnh phm: Cc loi ung th: ung th vm hng, ung th gan, ung th t cung, v cc bnh do virus, vi khun: virus HIV, vi khun than, vi khun t,... 3. Trong cng nghip: Sn xut protein ti t hp, sn xut axit amin, sn xut cc chng vi sinh vt dng trong cng nghip 4. Trong nng nghip Sinh vt bin i gen: cy trng bin i gen, ng vt bin i gen Ch phm bo v thc vt 5. Trong x l mi trng : X l nhim mi trng 1. 2. 3. 4. 5. 6. b.

Cng ngh vt liu mi Xu hng k thut gen: thuc cha bnh, bo v v nng cao sc khe con ngi Vt liu sinh hc Ci bin vt nui cy trng to cc ngun thc phm mi. 2. Gen l on Dna hoc Rna mang thng tin di truyn xc nh cu trc chui polypeptid hoc mt loi RNA. Ty theo mi loi gen c chc nng khc nhau , ch yu l coding genes m ha cho cc loi protein v noncoding gen. a.Gen m ha protein c chc nng: cu to c th, min dch, iu ha, bo v c th, sinh sn v di truyn, thch nghi, ... b. Gen m ha cc loi RNA: chc nng tng hp cc Rna, chc nng iu ha , chc nng bo v t bo c. Cc gen khng mang m: promoter, intron, telomer, cc trnh t lp Cu trc v sp xp gen vi eukaryote v prokaryote xem li di truyn hc. Cc phng php c bn trong k thut gen: Nguyn l: +Chun b gen cn tch dng +chn vector tch dng to vector ti t hp +Bin np vo vt ch +Sng lc cc dng t bo, nhn dng t bo ti t hp v nui cy +nui cy to mi trng sinh sn phm. - Phn bit vector tch dng: l cc phn t DNA c kch thc nh, cho php gn on DNA cn thit, c kh nng ti bn khng ph thuc vo s phn chia t bo, tn ti trong t bo vt ch v khng nh hng n b gen vt ch. Yu cu c nhiu im ct c hiu duy nht cho nhiu enzyme gii hn khc nhau ng thi mang gen tn hiu nhn bit. - Vector biu hin l vector tch dng c promoter mnh cho php biu hin gen ch th v gen tch dng to nn cc protein lai. Vector biu hin gm: promoter mnh, trnh t sao chp ori, v tr khi u phin m, v tr bm ca riboxome, tn hiu kt thc dch m, cc trnh t a ct ni v cc gen ch th. - Vector tch dng c th lm vec t biu hin nhng ngc li th cha chc Cc k thut chuyn gen: K thut chuyn gen trc tip: bin np, in xung, vi tim, sng bn gen, siu m Cc bc: 1. Chn t bo ch v vector mang gen cn biu hin. 2. Dng k thut a gen vo t bo ch 3. Sng lc chn la cc t bo bin np 4. Kim tra hot tnh. Gin tip: ti np, ________________________________ Phng php phn tch: PCR chun: phng php khuch i gen: 1. Bin tnh chung 94-95/5 2. Bt mi 50-60/2 3. Tng hp 72/3 Yu cu: thi gian u tho xon bin tnh chung g ht cc lin kt 95 /5 v thi gin sau n nh tng hp nt 72/10. Cc yu t chnh: khun, mi , dNTP, enzyme, cc yu t m khc. End: thu c DNA c hiu bt cp tng hp vi cp mi s dng PCR ngc: 1. Phin m ngc t mRNA thnh cDNA kp 2. Dng cDNA mch kp tin hnh PCR End : thu c DNA cha extron PCR lng: 1. PRC thng 2. Ly kt qu PCR ln 1 lm khun tip tc PCR ln 2: rt c hiu: ? tinh khit ca DNA End thu c DNA bn trong on DNA trn hoc chnh n m khng c DNA tp PCR o: Cu gen khi ch bit mt on trnh t: 1. Dng enzyme ct on trnh t bit, ni hai u trnh t nm hai u DNA mi. 2. S dng hai u bit trnh t thit k mi v PCR

End thu c DNA m ta mong mun khuch i mch di hn PCR phc: s dng vi dna ca sinh vt nhn chun do dna c nhiu intron: s dng mt hn hp mi: mi extron cn mt cp mi End thu c hn hp cc dna l extron hay ty thuc vo cc cp mi ta s dng. PCR ti ch: tin hnh c nh t bo trn lam knh, nh huyn dch v cc thnh phn cn cho pcr, tin hnh pcr v nhum quan st. End: khuch i v bit c v tr gen nm u ngi ta c th bit gen nm nst s my, nu cc t bo ln cn s dng my ct lt. Cc yu t nh hng: Tm 55-60 %GC 50-60 Len mi : 15-25bp Len sp: 450-500bp Mi bt cp c hiu kch thc khun, tinh sch ca khun Kch thc v hm lng mi Hm lng nu t do Ch nhit Enzyme bn nhit Dung dch m K thut in di v ly tm.K thut in di cho ta bit Dna da trn khi lng phn t. K thut lai phn t: nguyn tc: Tch v tinh sch DNA, s dng e gii hn ct thnh tng on nh in di trn gel agarose tch DNA theo kch thc Bin tnh Dna/gel bng Naoh Chuyn bn gel ln mng lai Thc hin ph lai vi mu d Kim tra kt qu bng fim nhy phng x Mu d l mch DNA n bit trnh t v nh du hunh quang hoc phng x. Cc k thut lai phn t : Souther n Blotting, Northern Blotting, Lai ti ch,

K thut s dng enzyme gii hn: 3 kiu c t u bng, ui 3 , ui 5. K thut DNA mng Tch dng v biu hin gen Chn ngun gen Chn vector Chn host cell Bin np gen Sng lc nui cy to dng thun mang gen tch dng Nghin cu iu kin gi v n nh Tch dng l chuyn gen ch vo t bo kh bin to cc dng t bo mang gen ch Tch dng n gen v tch dng a gen Chn gen Chn vector To vector ti t hp Bin np vector vo host cell d to chng ti t hp

Biu hin gen: iu ha biu hin gen pro: iu ha phin m, dch m, sau dch m. iu ha Eu: thay i cu trc gen, phin m, ct ni intron v tit mRNA, dch m ,phn hy mRNA, bin i hoc phn hy protein Vector biu hin l vec tor tch dng c promter mnh c kh nng biu hin c gen nh du v gen tch dng.