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1、引物合成常见问题（Commonproblemsinprimersynthesis）Publishedinthe2010-5-1311:33
2、onlyseetheauthor[reprint]primersynthesisisintroducedandthecommonproblemsprimerHowarethe1.primerssynthesized?Atpresent,thesynthesisofprimersadoptssolid-phasephosphitethreeestermethod
3、.TherearemanykindsofDNAsynthesis,aremainlyproducedbytheABI/PEcompany,nomatterwhatthemachinesynthesis,thesynthesisprincipleisthesame,themaindifferenceisthattheyieldlevelofdifferentamountofreagentconsumptionandsinglecycletime.Themainmodelsofsyntheticins
4、trumentShenergyGamblingcompanyABI3900isusedforhigh-throughputsynthesis,synthesisoflongchain1000MandPE8909mainlyusestheBeckmanDNAsynthesizer,primermodificationandhighODsynthesisbyABI394.ThesynthesisofDNAfragmentsbythemethodofphosphitethreeesterhasthech
5、aracteristicsofhighefficiency,rapidcouplingandrelativelystablestartingreaction.PhosphoramiditeestermethodisthreeDNAimmobilizedonDNAchainsynthesis,synthesisofthedirectionistobetheprimer3'endtothe5'endofsynthesis,theadjacentnucleotidesconnecting5'two'to
6、3byesterphosphatebond.Thefirststepistoreactwitha5-hydroxyprotectivegroupDMTandobtainafree5'-hydroxylgroupbythereactionofthenucleotideprotectedbytheactivegroupinthesolidcarrierCPGwiththethreechloroaceticacid;?secondstepsynthesisofDNArawmaterials,phosph
7、oramiditeprotectednucleotidemonomers,andtheactivatorwasmixed,nucleosidephosphiteintermediateshavebeenactivated,its3'endisactivated,5'-hydroxylstillprotectedbyDMT,condensationreactioninsolutionwithfree5'-hydroxyl.Thethirdstep,withcap?(capping)reaction,
8、condensationreactionmayhaveveryfew5'-hydroxylgroupdidnotparticipateinthereaction(lessthan2%),withaceticanhydrideand1-methylimidazoleterminatedbythereactions,theshortfragmentscanbeseparatedinthepurificationofoff.Inthefourthstep,undertheactionofoxidanti
9、odine,thephosphiteformschangeintoamorestablephosphoricacidthreeester.Aftertheabovefoursteps,adeoxyribonucleicacidisattachedtothenucleotideofthesolidphasecarrier.ThenremovetheprotectivegroupDMTonits5'hydroxylgroupwiththreechloroaceticacidandrep
