Mfundo yofunikira ya kaphatikizidwe ka DNA

Kaphatikizidwe kake ka DNA kaphatikizidwe kakapangidwe kagawo kolimba kaphatikizidwe kake ndi phosphoramidite chemistry.Mosiyana ndi kaphatikizidwe ka DNA yachilengedwe, zomwe zimapangidwa mu DNA synthesis ya DNA ndi DMT (4, 4-dimethoxytrityl) ndi phosphoramidite yosinthidwa deoxyriboxyside, monga momwe tawonetsera pa Chithunzi 1. Kuphatikizika kwa DNA kumachitika pakuthandizira kolimbaOligo Synthesis Column), mwachitsanzo CPG (galasi la pore loyendetsedwa) ndi PS (polystyrene), ndipo kaphatikizidwe konseko kamachitika paDNA/RNA synthesizer, zomwe ndi zida zathu zazikulu, zili ndi mitundu yosiyana monga: HY Single Channel Synthesizer, HY-12, HY-192 ndi zina, njira yophatikizira imachokera ku 3' mpaka 5 ', ndipo nucleotide imayambitsidwa ku chithandizo cholimba kudzera m'modzi. kaphatikizidwe kuzungulira.Nthawi zambiri kaphatikizidwe kaphatikizidwe kamakhala ndi masitepe anayi, Kuteteza, Kuphatikizika, Kuyika ndi Oxidation (Chithunzi 2).The Deprotection imayikidwa kuti ichotse gulu la DMT pa chithandizo cholimba kapena gulu la 5' hydroxyl pa nucleoside yapitayi, 3% trichloroacetic acid mu dichloromethane imagwiritsidwa ntchito ngati reagent deprotection.Ndiye phosphoramidite kusinthidwa deoxyriboxyside analoledwa kukhudzana hydroxyl gulu mothandizidwa ndi activator, mwachitsanzo 5-ethylthiotetrazole kapena 4, 5-dicyanoimidazole, kupanga phosphitetriester (III), ndipo anazindikira Coupling sitepe.Gawo la Capping limachitika kuti aletse gulu lomwe silinachitepo kanthu la hydroxyl panthawi yolumikizana, kuti muchepetse kupangika kwamayendedwe osafunikira.Pomaliza, phosphitetriester yosakhazikika (III) imapangidwa ndi oxidized to chemical stable phosphortriester (V) yokhala ndi ayodini monga oxidant mu pyridine.

DNA yopangidwa ikhoza kupatulidwa kuchokera ku chithandizo cholimba ndi aminolysis, gulu lotetezedwa la 2-cyanoethyl pa phosphortriester ndipo amide pa nucleobase imadulidwa nthawi yomweyo, Synthesis Plates ndi Synthesis Columns muzitsulo zimayikidwa mwachindunji mu chipinda chochitira. chaZida Zoteteza.DNA Crude ikhoza kukonzedwa ndi HPLC, electrophoresis ndi OPC pakufunika, titha kukupatsaninsoZida Zoyeretseraza Post-processing.