Ụkpụrụ bụ isi nke njikọ DNA

Ngwakọta DNA nke kemịkalụ dabere na atụmatụ njikọ siri ike na kemịkalụ phosphoramite.Dị iche na njikọ DNA nke ndu, ihe dị na njikọ DNA nke kemịkalụ bụ DMT (4, 4-dimethoxytril) na phosphoramidite gbanwetụrụ deoxyriboxyside, dị ka e gosiri na Figure 1. A na-eme njikọ DNA na nkwado siri ike (anyị nwere ike ịnye ụdị dị iche iche).Ogidi Oligo Synthesisie CPG (achịkwa pore iko) na PS (polystyrene), na dum njikọ a rụrụ na a.DNA/RNA synthesizer, nke bụ isi akụrụngwa anyị, nwere ụdị dị iche iche dị ka: HY Single Channel Synthesizer, HY-12, HY-192 na wdg, ntụziaka njikọ sitere na 3' ruo 5', na nucleotide na-ewebata nkwado siri ike site na otu. okirikiri njikọ.Usoro usoro njikọ na-enwekarị usoro anọ, Deprotection, Coupling, Capping and Oxidation (Figure 2).Edebere Nchekwa ahụ iji wepụ otu DMT na nkwado siri ike ma ọ bụ otu 5' hydroxyl na nucleoside gara aga, a na-eji 3% trichloroacetic acid dị na dichloromethane dị ka ihe nchebe.Mgbe ahụ phosphoramidite gbanwetụrụ deoxyriboxyside ka e kwere ka ikpughe hydroxyl otu site n'enyemaka nke activator, ya bụ 5-ethylthiotetrazole ma ọ bụ 4, 5-dicyanoimidazole, na-akpụ phosphitetriester (III), na ghọtara nzọụkwụ njikọ.A na-eme nzọụkwụ capping iji gbochie ìgwè hydroxyl na-emeghị eme n'oge njikọ njikọ, iji belata nhazi usoro nrụrụ na-achọghị.N'ikpeazụ, phosphitetriester na-adịghị akwụsi ike (III) bụ oxidized na phosphortriester kwụsiri ike (V) na ayodiin dị ka oxidant na pyridine dị ugbu a.

DNA synthesized nwere ike ikpochapụ site na nkwado siri ike site na aminolysis, otu 2-cyanoethyl na-echebe na phosphortriester na amide na nucleobase na-agbaji n'otu oge ahụ, a na-etinye Plates Synthesis na Synthesis Columns na racks ozugbo na ụlọ mmeghachi omume. nkeNgwa nchekwa.Enwere ike ịkwadebe DNA crude site na HPLC, electrophoresis na OPC na-achọ, anyị nwekwara ike ịnye gịAkụrụngwa dị ọchamaka Post-processing.