Umgaqo osisiseko we-DNA synthesis

I-chemical DNA synthesis isekelwe kwisicwangciso esiqinileyo se-synthesis kunye ne-phosphoramidite chemistry.Ihluke kwi-biological DNA synthesis, izinto kwi-chemical DNA synthesis yi-DMT (4, 4-dimethoxytrityl) kunye ne-phosphoramidite eguqulweyo ye-deoxyriboxyside, njengoko kubonisiwe kuMfanekiso 1.IKholam yokwenziwa kweOligo), oko kukuthi, i-CPG (iglasi yepore elawulwayo) kunye ne-PS (ipolystyrene), kwaye yonke i-synthesis yenziwa kwiDNA/RNA synthesizer, eyona zixhobo zethu eziphambili, zinemodeli eyahlukileyo efana ne: HY Single Channel Synthesizer, HY-12, HY-192 kunye njl njl, ulwalathiso lwe-synthesis luvela kwi-3 'ukuya ku-5', kwaye i-nucleotide ifakwe kwinkxaso eqinile ngenye. umjikelo wokwenziwa.Umjikelo oqhelekileyo we-synthesis uqukethe amanyathelo amane, ukuKhuselwa, ukuPhathwa, ukuCapping kunye ne-Oxidation (Umfanekiso 2).I-Deprotection isetelwe ukususa iqela le-DMT kwinkxaso eqinile okanye iqela le-5 'hydroxyl kwi-nucleoside yangaphambili, i-3% ye-trichloroacetic acid kwi-dichloromethane isetyenziswa njenge-reagent yokukhusela.Ke phosphoramidite modified deoxyriboxyside wavunyelwa ukuba exposure hydroxyl iqela ngoncedo activator, oko kukuthi 5-ethylthiotetrazole okanye 4, 5-dicyanoimidazole, ukwenza phosphitetriester (III), kwaye waqonda inyathelo Coupling.Inyathelo le-Capping lenziwa ukuvimba iqela le-hydroxyl elingaphendulwanga ngexesha lokudibanisa, ukunciphisa ukwakheka kolandelelwano lwesiphako esingafunekiyo.Ekugqibeleni, i-phosphitetriester engazinzanga (III) i-oxidized kwi-chemical stable phosphortriester (V) kunye ne-iodine njenge-oxidant kwi-pyridine yangoku.

I-DNA edibeneyo ingaba yi-cleavage kwinkxaso eqinile nge-aminolysis, iqela elikhuselweyo le-2-cyanoethyl kwi-phosphortriester kunye ne-amide kwi-nucleobase ihlanjululwe ngexesha elifanayo, i-Synthesis Plates kunye ne-Synthesis Columns kwii-racks zifakwe ngokuthe ngqo kwigumbi lokuphendula. yeIzixhobo zokuKhusela.I-DNA ekrwada inokulungiswa yi-HPLC, i-electrophoresis kunye ne-OPC xa ifunwa, sinokukunikaIzixhobo zokucocayePost-processing.