ʻO ke kumu kumu o ka synthesis DNA

Hoʻokumu ʻia ka synthesis DNA kemika i ka hoʻolālā synthesis solid-phase a me ke kemika phosphoramidite.ʻOkoʻa mai ka biological DNA synthesis, ka mea i loko o ka DNA synthesis kemika ʻo DMT (4, 4-dimethoxytrityl) a me ka phosphoramidite deoxyriboxyside i hoʻololi ʻia, e like me ka mea i hōʻike ʻia ma ka Figure 1. Hoʻokō ʻia ka synthesis DNA ma ke kākoʻo paʻa (hiki iā mākou ke hāʻawi i nā ʻano like ʻole.Oligo Synthesis Column), ʻo ia hoʻi ʻo CPG (ke aniani pore controlled) a me PS (polystyrene), a ua hana ʻia ka synthesis holoʻokoʻa ma kahiDNA/RNA synthesizer, ʻo ia kā mākou mea hana nui, he kumu hoʻohālike like ʻole e like me: HY Single Channel Synthesizer, HY-12, HY-192 a me nā mea ʻē aʻe, ʻo ke kuhikuhi synthesis mai 3 'a 5', a ua hoʻokomo ʻia kahi nucleotide i ke kākoʻo paʻa ma o hoʻokahi. pōʻaiapili synthesis.He ʻehā mau ʻanuʻu ka pōʻaiapili synthesis maʻamau, Deprotection, Coupling, Capping and Oxidation (Figure 2).Hoʻonohonoho ʻia ka Deprotection e wehe i ka pūʻulu DMT ma ke kākoʻo paʻa a i ʻole ka hui 5' hydroxyl ma ka nucleoside mua, hoʻohana ʻia ka 3% trichloroacetic acid i ka dichloromethane ma ke ʻano he deprotection reagent.A laila ua ʻae ʻia ka phosphoramidite deoxyriboxyside i hoʻololi ʻia e hōʻike i ka hui hydroxyl me ke kōkua o activator, ʻo ia hoʻi 5-ethylthiotetrazole a i ʻole 4, 5-dicyanoimidazole, e hana ana i ka phosphitetriester (III), a hoʻomaopopo i ka ʻanuʻu Coupling.Hana ʻia kahi ʻanuʻu Capping e ālai i ka pūʻulu hydroxyl i hoʻokō ʻole ʻia i ka wā o ka hoʻohui ʻana, e hōʻemi i ka hoʻokumu ʻana o nā kaʻina hemahema i makemake ʻole ʻia.ʻO ka hope loa, ua hoʻokahe ʻia ka phosphitetriester paʻa ʻole (III) i ka phosphotriester paʻa kemika (V) me ka iodine ma ke ʻano he oxidant i kēia manawa o pyridine.

Hiki ke hoʻokaʻawale ʻia ka DNA synthesized mai ke kākoʻo paʻa e aminolysis, ʻo ka 2-cyanoethyl protected group ma ka phosphotriester a me ka amide ma ka nucleobase i hoʻokaʻawale ʻia i ka manawa like, ua kau pololei ʻia nā Synthesis Plates a me Synthesis Columns i loko o ka keʻena pane. o kaLako Hoopae.Hiki ke hoʻomākaukau ʻia ka Crude DNA e HPLC, electrophoresis a me OPC ma ke koi, hiki iā mākou ke hāʻawi iā ʻoe i kaLako Hoomaemaeno ka hana hope.