Lub nucleic acid synthesis yog ua los ntawm cov khoom theem sialic amide triglyceride txoj kev, uas yog 3' kawg ntawm DNA yog immobilized ntawm ib tug khoom theem substrate thiab nucleotides ntxiv nyob rau hauv lub 3′ mus rau 5′ kev taw qhia kom txog rau thaum lub xav DNA fragment yog synthesized. .Qhov no txawv ntawm DNA synthesis los ntawm daim ntawv thov ntawm DNA polymerase.
Lub 3' kawg ntawm thawj lub hauv paus yog immobilized ntawm CPG thaum lub sij hawm synthesis, lub 5'-OH ntawm lub hauv paus tom ntej yog tiv thaiv nrog di-p-tolyl trityl DMT, cov amino pawg ntawm lub hauv paus yog tiv thaiv nrog benzoic acid thiab 3' -OH tom qab ntawd qhib nrog cov amino phosphate compound.1 lub hauv paus ntawm 5 5'-OH ntawm 1 lub hauv paus thiab 3'-OH ntawm lub hauv paus tom ntej tsim ib qho phosphite triglyceride, uas yog tom qab ntawd oxidised nrog iodine rau phosphate triglyceride, tiv thaiv ntawm lub hauv paus thib ob 5'-OH raug tshem tawm los ntawm ntxiv dichloroacetic acid DM cycles los ntawm kev sib ntxiv ntawm lub hauv paus tom ntej, thiab tom qab synthesis tus tiv thaiv ntawm 5'-OH yog tshem tawm nrog ib tug qaug zog acid Lub fragment yog disconnected los ntawm cov khoom resin nrog concentrated ammonium hydroxide, tus tiv thaiv raug tshem tawm ntawm lub hauv paus. nrog cov concentrated ammonium hydroxide nyob rau hauv cov cua kub, cov ammonium hydroxide raug tshem tawm, qhov tawg yog lub tshuab nqus tsev qhuav, thiab cov nucleic acid yog purified los ntawm cov kua chromatography los yog PAGE.
Cov kauj ruam ntawm Oligo Synthesis
Deblocking
Cov5 kawg DMT tiv thaiv pab pawg raug tshem tawm los ntawm kev ntxiv TCA tshuaj.
Ua kom
Sib tov cov activator nrog oligonucleotide monomer los tsim cov active oligonucleotide monomer intermediate.
Kev sib txuas
5-terminal hydroxyl pab pawg reacts nrog cov nquag oligonucleotide nruab nrab hauv cov tshuaj tiv thaiv kev sib xyaw ua ke los tsim cov phosphite triglyceride tsis ruaj khov.
Capping
Qhov sib ntxiv ntawm tus neeg sawv cev capping ua rau acetylation capping cov tshuaj tiv thaiv nrog ntau pawg hydroxyl uas tsis koom nrog cov tshuaj tiv thaiv condensation.
Oxidation
Qhov sib ntxiv ntawm iodine tov uas muaj dej reacts nrog unstable phosphite bonds los ntawm oxidation los tsim ruaj khov phosphoric acid triglyceride bonds.
Cyanoethyl Cleavage
Cov khoom lag luam nucleic acid tom qab kev sib txuas, nrog rau kev sib ntxiv ntawm DEA daws kom tshem tawm cov pab pawg cyanoethyl los ntawm phosphate triglyceride daim ntawv cog lus.
Cleaved & Deprotection
Cov nucleic acid fragments yog cleaved los ntawm cov khoom theem thauj khoom los ntawm kev ntxiv cov concentrated ammonium hydroxide tov thiab ces rhaub kom tshem tawm cov pab pawg tiv thaiv los ntawm hydrogen bonds ntawm lub hauv paus.
Post lub sij hawm: Nov-21-2022