Mfundo za Nucleic Acid Synthesis

Kuphatikizika kwa nucleic acid kumachitika pogwiritsa ntchito njira yolimba ya sialic amide triglyceride, pomwe ma 3'mapeto a DNA sasunthika pagawo lolimba la gawo lapansi ndipo ma nucleotides amawonjezedwa munjira ya 3' mpaka 5' mpaka chidutswa cha DNA chomwe chimafunidwa chitapangidwa. .Izi zimasiyana ndi kaphatikizidwe ka DNA pogwiritsira ntchito DNA polymerase.

Mapeto a 3 a maziko oyambirira ndi osasunthika pa CPG panthawi ya kaphatikizidwe, 5'-OH ya maziko otsatirawa amatetezedwa ndi di-p-tolyl trityl DMT, gulu la amino pamunsi limatetezedwa ndi benzoic acid ndi 3′. -OH ndiye adamulowetsa ndi amino phosphate pawiri.Mtsinje umodzi wa 5 5'-OH wa 1 maziko ndi 3'-OH wa maziko otsatirawa amapanga phosphite triglyceride, yomwe imayikidwa ndi ayodini ku phosphate triglyceride, wotetezera pa maziko achiwiri 5'-OH amachotsedwa ndi kuwonjezera dichloroacetic acid DM cycles kudzera kuwonjezera pa maziko otsatirawa, ndipo pambuyo kaphatikizidwe chitetezo pa 5'-OH amachotsedwa ndi asidi ofooka Chidutswacho chimachotsedwa ku utomoni wolimba ndi concentrated ammonium hydroxide, wotetezera amachotsedwa pamunsi. ndi ndende ya ammonium hydroxide pansi pa kutentha, ammonium hydroxide imachotsedwa, chidutswacho chimauma, ndipo nucleic acid imayeretsedwa ndi madzi chromatography kapena PAGE.

Masitepe a Oligo Synthesis

Kutsekereza

TheGulu loteteza la 5 la DMT limachotsedwa powonjezera yankho la TCA.

Kutsegula

Kusakaniza activator ndi oligonucleotide monoma kupanga yogwira oligonucleotide monoma wapakatikati.

Kulumikizana

Gulu la 5-terminal hydroxyl limachita ndi oligonucleotide yogwira ntchito yapakatikati mu anti-generative condensation reaction kuti apange chomangira chosakhazikika cha phosphite triglyceride.

Capping 

Kuphatikizika kwa capping agent kumapangitsa acetylation capping reaction ndi magulu ochulukirapo a hydroxyl omwe sakhudzidwa ndi condensation reaction.

Kuchuluka kwa okosijeni 

Kuphatikizika kwa ayodini yankho lomwe lili ndi madzi limakhudzidwa ndi zomangira zosakhazikika za phosphite ndi okosijeni kuti apange zomangira zokhazikika za phosphoric acid triglyceride.

Cyanoethyl Cleavage

The nucleic acid mankhwala pambuyo kaphatikizidwe, ndi kuwonjezera kwa DEA yankho kuchotsa cyanoethyl gulu ku phosphate triglyceride chomangira.

Kutsekedwa & Kutetezedwa

Zidutswa za nucleic acid zimang'ambika kuchokera ku chonyamulira cholimba powonjezerapo yankho lokhazikika la ammonium hydroxide ndikutenthedwa kuti achotse magulu oteteza kumabondi a haidrojeni a mazikowo.