cau 9 : Khi co cac doan ADN hpac c ADn mong muon . Buoc tiepo theo la gan chung vao vector chuyen gen de tap plasmid tai to hop . CO 3 pp chjnh :

a, Su dung dau dinh : Vctor chuyen gen la ADN vong tron dc cat boi 1 loai restritoion enzyme ( Eco RI ) chuyen thanh dang thag co 2 dau co ket. Cac ftu ADN cua bo gen khac cung dc cat boi cung 1 loai enzy,e gioi han nay se tao ra nhieu nhieu doan ADN thang co cac dau co ket. TRon lan ADn la voi vector chuyen gen da bi cat bang cung 1 loai enzyme gioi han tren. Cac dau co ket cua 2 loai ADN la va vector bo sung se bat cap voi nhau . , Enzyme ligase se noi ADN la voi ADN cua vector lai voi nhau  bang cau noi phosphodiester tao nen ftu ADN tai to hop . PP nay dc su dung rong rai de lap ngan hang gen cua 1 sv.

b , pp DUng doan noi :

Cac doan noi la cac doan oligonucleotitde co chieu dai  tu 10- 20 nucleotit dc tong hop hoa hoc .Trong moi doan linker fai mag trinht u nhan biet dac hieu cua 1 restriction enzyme nao do. Sau do dung enzyme DNA ligase cua phage T4 noi cac doan nay lai vs nhau . Cac doan noi se gan vao 2 dau cua doan ADN la . va khi cat bang enzyme gioi han  ( VD : EcorRI )  se tao 2 dau co ket cua EcoRI . NHo do ma ADN la co the gan vao vector chuyen gen cung bi cat boi EcoRI  mac du trc do ban than ADN la k co trinh tu nhan biet dac hieu doi vs enzyme gioi han . PP nay thuong dc dung trong truong hop ADN la  va vector chuyen gen  k co cung trinh tu nhan biet cua enzyme gioi han

VD : mach kep c ADN cua cac doan ADN co dau bang .

c, PP dung enzyme terminal transferiase

- PP nay dua vao kha nang dac biet cua enzyme terminal nucleotidyl transferase, co the gan cung 1 loai nucleotide thanh chuoi  ( homo polymer ) vao dau mut 3'OH  cua mach ADN .

- Theo mo ta , vectoer chuyen gen va ADN la can gan dc cat boi  exonuclease hoac enzyme gioi han ( VD : PstI ) . Sau do vector chuyen gen dc u vs enzyme  terminal transferase va 1loai nucleotide ( VD : dGTP ) ket qua tap thanh o 2 dau mut 3' OH cua vector 1 doan poly G ( 3'GGGGG) Doan ADN la sau khi cat boi exonuclease cung dc u vs enzyme  terminal transferae va bo sung vao moi truong u la dCTP de tao duoi 3'CCCCC bo sung vs doan GGGGG cua vector . Nho do ma ADN la co the gan vao vector do bat cap bo sung giua G va C . Gai doan cuoi cung  la dung enzyme ADN poly me rase I gan cac nuleotide tuong ung vao cho trong va ligase han dicnh ADN la voi vector tao nen ADN tai to hop . Voi pp nay co the gan ADN la vao bat ki vector chuyen gen nao .