Multiplex PCR System Optimization with Potato SSR Markers.pdfVIP

一 ● · September2012 Vo1．19 No．3 20—27 ’：：，ScienceDirect Availableonlineatw、f ．sciencedirect．com JournalofNortheastAgriculturalUniversity(EnglishEdition) MultiplexPCRSystem OptimizationwithPotatoSSRMarkers W angShao-peng，LiuShangwu，LiYong，LiuW ei—ring，andLvDian。qiu viruses—freeSeedlingResearchInstitute，HeilongjiangAcademyofAgriculturalSciences，HeilongiiangPotatoEngineeringandTechnologyR。。。。h Center，Harbin 150086，China Abstract：PotatovarietyKexinl8wasusedastestingmaterialsinthisresearchtostudytheinfluenceonmaincomponentsin multip1exPCR system，differentprimerratiosandannealingtemperaturesinSSRmarkeramplification．Concentrationandgradient exDerimentsf-orfourcomponents(enzyme，MgC12，DNA templateanddNTPs)inPCRsystemwereusedintheresearchwiththe concentrationoftheothercomponentremainedthesame；theorthogonaldesignL9(34)wasappliedintheoptimizationoffoursetsof DrimersfSTM0014，Pat，SSI，andUGP)inthereactionsystem atthreelevels；thetemperaturegradientselectionwasusedtofindout theopthnum annealingtemperaturefortheprimer．TheoptimizedmultiplexPeR system ofpotatoSSRmarkerwiht atotalvolumeof 20gL：2．5gL25mmo1．L～MgCI2，0．6gL 10mmol·L～dNTPs，0．8U Taq，80ngDNA templatewasultimatelyestablishedthrough thecomparisonandanalysisoftestresults；theratiooffourpairsof4mmolL‘ primerswas2：1：2：3，andtheannealingtemperature was54．7℃ ．Theoptimizedreactionsystem couldberepeatedstably；andthestableandreliableamplificationresultswereableto c1earlvdistinguishdifferentpotatovarieties．Thisresearchbuiltthesolidfoundationofrthefurtherstudyofgeneticdiversityofpotato germplasm sandconstructionofDNA fingerprinting．． Keywords：potato，SSRmarker，multiplexPCRsy