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Nguyn Ngc Phc CNSH- K32 Cc bc to dng phn t: To dng phn t dng k thut di truyn phn lp v lm thun mt gene

e v gm cc bc c bn sau Bc 1: Tch v phn on DNA ngun bng enzyme ct hn ch. Bc 2: Gn cc on DNA b ct trn vo cc vector hoc plasmid to dng. Bc 3: Bin np vector ti t hp vo vi khun hoc t bo vt ch v gi vector ti t hp trong t bo vt ch, Bc 4: To dng, pht hin v lm thun dng mc tiu. Bc 5: Chn lc dng c gene cn khuch i v nui cy dng mc tiu ly trch v nghin cu DNA ti t hp.

Bc 1: Sau khi tch c mt lng t bo nht nh th ta tin hnh ct hay phn on gene cn to dng bng enzyme ct hn ch. Ty vo trnh t ca gene ch m ta la chn enzyme ct hn ch ph hp. Mi enzyme hn ch nhn bit v ct mt trnh t DNA c trng cha 4 hoc 4 cp nucleotide. V d: enzyme EcoRI chit t E.coli ct trnh t GAATTC, enzyme TaqI ca Thermus aquaticus ct trnh t TCGA. V mt enzyme hn ch nhn bit mt trnh t duy nht, cho nn s v tr ct trn mt phn t DNA c bit thng rt nh.Cc on DNA ct bi enzyme hn ch s c u bng hoc u sole v c th phn tch theo kch thc bng in di agarose gel nghin cu.

Sau khi ct bng enzyme hn ch thch hp th ta c mt s lng ln cc on gene c trnh t khc nhau. Bc 2: Bc ny ta tin hnh gn cc on DNA thu c trn s c gn ng ha tr in vitro vo vector to dng bng cch dng enzyme gn ligase.Trc khi gn, vector c ct ra mt v tr n, thng thng l cng vi enzyme c dng ct DNA ngun, nhm to iu kin kt dnh b tr cho cc u ca on DNA chn vo. Hai loi vector c s dng ph bin nht l mt loi c ngun gc t cc plasmid ca vi khun v mt loi khc c ngun gc virus xm nhim vo t bo vi khun (phage). Cc plasmid l nhng phn t DNA mch vng, si i, kch thc t 1200kb, kich thc tng i nh, mang nhiu gn ch th chn lc cho php xc nh cc nhn t bin np v duy tr plasmid trong qun th vi khun. Cc plasmid uc dng ph bin nht hin nay l cc plasmid a nng v thuc th h th 3.

Ty vo chiu di ca on gene m mnh cn gn m ta chn plasmid gn hay chn phage, v phage vector (phage ) mang c on DNA ln hn (15-23 kb) so vi plasmid, d bo qun v d tch ra phn tch. Bc 3: Bin np vector ti t hp vo vi khun hoc t bo vt ch. Sau khi to c vector ti t hp mang gene ngoi lai, vic tip theo l bin np n vo t bo vt ch.T bo vt ch thng c s dng l vi khun E.coli khuch i mt lng ln DNA ca plasmid ti t hp cho cc phn tch v sau. Hai phng php c dng bin np vector ti t hp vo E.coli l in bin np v ha bin np. Ty vo u im v nhc im ca mi phng php m ta chn phng php thch hp chuyn vector ti t hp vo t bo vt ch. Mc ch l tng hiu qu ca vic bin np. Bc 4: Sau khi bin np vector ti t hp vo vi khun hoc t bo vt ch th ta phi to iu kin cho th ti t hp ny pht trin. Ni cch khc th phi cho gene a vo c phin m, dch m v biu hin ra bn ngoi. DNA c to dng trong plasmid sn xut ra cc khun lc khi cc nui cy bin np c dn mng trn a agar cha mi trng sinh trng v nui cy di nhng iu kin thch hp. Chng ta c th da v cc khun lc to vt tan hay c khng khng sinh hay khng phn lp v chn ra khun lc mang gene mnh cn to dng. Da vo cc phng php khc nhau ta nhn bit c khun lc no mang vector ti t hp m mnh quan tm, t chng ta ly ra em i phn lp, in di ct ly on gene m mnh mun to dng. Nhm lm thun khit dng mc tiu. Bc 5: sau khi tch chit c on gene mnh quan tm th ta c th tin hnh PCr tng s lng on gene ny ln. Hoc nui cy cc t bo cha cc gene mnh cn quan tm tng s lng v nghin cu cu trc v sn phm do gene ny to ra. ng dng ca vic to dng gene: Cng ngh sinh hc s dng cc t bo sng to ra cc nguyn liu hu dng cho con ngi: + Nm men c dng xn xut bia, ru vang..

+ Cc vi khun c dng sn xut cc cht khng sinh, ru v mt s cn phm khc. S to dng gene c dng sn xut mt lng ln protein. Phn ln cc gene c th c chn vo vi khun hoc nm men. Cc t bo ny c th c dng to ra mt lng ln sn phm. Vic to dng gene gip to ra cc ging cy trng cho nng sut cao (la chuyn gene) v tng kh nng chng chu vi iu kin mi trng so vi ging ban u. Vic to dng gene c th gip to ra cc ging cy trng chuyn gene c mang vaccine ti t hp, m thay v ta tim hay ung vaccine th by gi ta c th n sn phm ca cy chuyn gene. Gip gim chi phi sn xut cho ngnh y dc v gi thnh sn phm gim. C mt s sn phm nh hormone, thuc khng sinh nu sn xut theo phng php truyn thng th rt mt thoi gian v gi thnh sn phm th cao. Thng qua vic to dng gene chuyn gene cn quan tm hay m ha cc protein quan tm vo trong mt loi sinh vt c tc tng hp sn phm nhanh hn th gi thnh sn phm s r hn ( insulin, interleukin ) . Ngoi ra to dng gene cn c ng dng trong vic x l mi trng, gip gim bt nhim.

Chng ta phi to dng gene khi gene mnh cn quan tm khng bit c cu trc, di ngn nhu th no. ng thi s lng gene quan tm rt t nn ta phi tin hnh clone n ln c s lng thichs hp phc v cho mc ch nghin cu v cc mc ch khc na.