Isimiso esiyisisekelo se-DNA synthesis

I-chemical DNA synthesis isuselwe ku-solid-phase synthesis strategy kanye ne-phosphoramidite chemistry.Ihlukile ekuhlanganiseni kwe-DNA yebhayoloji, okokusebenza kukhemikhali ye-DNA synthesis yi-DMT (4, 4-dimethoxytrityl) kanye ne-phosphoramidite modified deoxyriboxyside, njengoba kukhonjisiwe kuMfanekiso 1. Ukuhlanganiswa kwe-DNA kwenziwa ngokusekela okuqinile (singanikela ngezinto ezihlukahlukeneIkholomu ye-Oligo Synthesis), okungukuthi i-CPG (ingilazi yezimbotshana elawulwayo) kanye ne-PS (i-polystyrene), futhi konke ukuhlanganisa kwenziwaI-DNA/RNA synthesizer, okuyimishini yethu eyinhloko, inemodeli ehlukile efana nale: HY Single Channel Synthesizer, HY-12, HY-192 nokunye, isiqondiso se-synthesis sisuka ku-3' kuya ku-5', futhi i-nucleotide yethulwa ekusekelweni okuqinile ngeyodwa. umjikelezo wokuhlanganisa.Umjikelezo ojwayelekile wokuhlanganisa uqukethe izinyathelo ezine, Ukuvikela, Ukuhlanganisa, Ukuhlanganisa kanye Ne-Oxidation (Umfanekiso 2).I-Deprotection isethelwe ukususa iqembu le-DMT kusekelo eliqinile noma iqembu le-5' hydroxyl ku-nucleoside yangaphambilini, i-3% trichloroacetic acid ku-dichloromethane isetshenziswa njengesisetshenziswa sokuvikela.Khona-ke i-phosphoramidite modified deoxyriboxyside yavunyelwa ukuchayeka eqenjini le-hydroxyl ngosizo lwe-activator, okungukuthi i-5-ethylthiotetrazole noma i-4, i-5-dicyanoimidazole, eyenza i-phosphitetriester (III), futhi yaqaphela isinyathelo sokuhlanganisa.Isinyathelo se-Capping senziwa ukuvimba iqembu le-hydroxyl elingaphenduliwe ngesikhathi sokuhlanganisa, ukunciphisa ukwakheka kokulandelana kokukhubazeka okungafuneki.Ekugcineni, i-phosphitetriester engazinzile (III) ifakwe i-oxidized ku-phosphortriester yamakhemikhali ezinzile (V) ene-iodine njenge-oxidant okwamanje ye-pyridine.

I-DNA ehlanganisiwe ingase ihlukane ekusekelweni okuqinile nge-aminolysis, iqembu elivikelwe i-2-cyanoethyl ku-phosphortriester futhi i-amide ku-nucleobase ihlukaniswa ngesikhathi esifanayo, ama-Synthesis Plates kanye namakholomu e-Synthesis kuma-rack afakwa ngqo ekamelweni lokuphendula. kweIzinsiza Zokuvikela.I-DNA engahluziwe ingalungiswa yi-HPLC, i-electrophoresis ne-OPC uma idingeka, singakunikeza futhiIzinsiza Zokuhlanzayokucubungula ngemuva.