Ilana ipilẹ ti iṣelọpọ DNA

Iṣajọpọ DNA ti kẹmika da lori ilana imudara-alakoso ti o lagbara ati kemistri phosphoramidite.Yatọ si iṣelọpọ DNA ti ibi, ohun elo ti o wa ninu iṣelọpọ DNA kemikali jẹ DMT (4, 4-dimethoxytril) ati phosphoramidite ti a yipada deoxyriboxyside, gẹgẹ bi o ti han ni Nọmba 1. A ṣe agbekalẹ DNA kolaginni lori atilẹyin to lagbara (a le funni ni ọpọlọpọ awọn tiỌwọn Synthesis Oligoie CPG (dari pore gilasi) ati PS (polystyrene), ati gbogbo kolaginni ti wa ni ṣe lori kanDNA/RNA synthesizer, eyiti o jẹ ohun elo akọkọ wa, ni awoṣe ti o yatọ gẹgẹbi: HY Single Channel Synthesizer, HY-12, HY-192 ati bẹbẹ lọ, itọsọna iṣelọpọ lati 3 'si 5', ati pe nucleotide ti ṣe afihan si atilẹyin to lagbara nipasẹ ọkan. ọmọ kolaginni.Yiyipo iṣelọpọ igbagbogbo ni awọn igbesẹ mẹrin, Idabobo, Isopọpọ, Capping ati Oxidation (Aworan 2).A ṣeto Idabobo lati yọ ẹgbẹ DMT kuro lori atilẹyin to lagbara tabi ẹgbẹ 5' hydroxyl lori nucleoside iṣaaju, 3% trichloroacetic acid ni dichloromethane ni a lo bi reagent idabobo.Lẹhinna phosphoramidite ti a yipada deoxyriboxyside ti gba laaye si ẹgbẹ ifihan hydroxyl pẹlu iranlọwọ ti activator, ie 5-ethylthiotetrazole tabi 4, 5-dicyanoimidazole, ti o ṣẹda phosphitetriester (III), ati pe o rii igbesẹ Isopọpọ.Igbesẹ Capping kan ni a ṣe lati ṣe idiwọ ẹgbẹ hydroxyl ti ko ni idahun lakoko igbesẹ isọpọ, lati dinku dida awọn ilana abawọn aifẹ.Nikẹhin, phosphitetriester ti ko ni iduroṣinṣin (III) jẹ oxidized si kemikali iduroṣinṣin phosphortriester (V) pẹlu iodine bi oxidant ni bayi ti pyridine.

DNA ti a ti ṣajọpọ le jẹ fifọ lati atilẹyin to lagbara nipasẹ aminolysis, ẹgbẹ ti o ni aabo 2-cyanoethyl lori phosphortriester ati amide lori nucleobase ti pin ni akoko kanna, Awọn awopọ Synthesis ati Awọn ọwọn Synthesis ni awọn agbeko ti wa ni gbe taara ni iyẹwu ifura. ti awọnAwọn ohun elo Idaabobo.DNA robi le jẹ pese sile nipasẹ HPLC, electrophoresis ati OPC lori ibeere, a tun le pese fun ọ niOhun elo ìwẹnumọfun Post-processing.