资源描述：
《Genome Res.-1994-Edwards-S65-75》由会员上传分享，免费在线阅读，更多相关内容在学术论文-天天文库。
1、Downloadedfromgenome.cshlp.orgonNovember17,2016-PublishedbyColdSpringHarborLaboratoryPressll
2、llllManualSupplementMultiplexPCR:EversinceitwasshownthatPCRcouldsimultaneouslyamplifymultiplelociinthehumandystrophingene,(1)multiplexPCRhasbeenfirmlyestablishedAdvantages,asageneraltechnique.Ashortlisto
3、fmultiplexPCRapplicationsnowin-Development,cludespathogenidentification,genderscreening,linkageanalysis,forensicstudies,templatequantitation,andgeneticdiseasediagnosis.MultiplexPCRandcanbeatwo-ampliconsystemoritcanamplify13ormoreseparateregionsApplicationsofDNA.Itmaybetheendpointofanalysis,orpre
4、liminarytofurtheranalysessuchassequencingorhybridization.ThestepsfordevelopingamultiplexPCRandthebenefitsofhavingmultiplefragmentsamplifiedsimultaneously,however,aresimilarineachsystem.ThisreviewfocusesonmultiplexsystemsinwhicheachprimerpairtargetsMaryC.Edwardsandasinglelocus,unlikeRAPD(2)andalu
5、morph{3)PCRreactions,whichamplifyRichardA.Gibbsmultiplelociwithasingleprimerorprimerset.InstituteforMolecularGenetics,BaylorCollegeofMedicine,ADVANTAGESOFMULTIPLEXPCRHouston,Texas77030InternalControlsPotentialproblemsinPCRincludefalsenegativesduetoreactionfailureorfalsepositivesduetocontaminatio
6、n.Falsenegativesareoftenrevealedinmultiplexamplificationbecauseeachampliconprovidesaninternalcontrolfortheotheramplifiedfragments.Forexample,multipleexonsmaybeam-plifiedinassaysthatsurveyforgenedeletion.UnlesstheentireregionscannedbythemultiplexPCRisdeleted,amplificationofsomefragment(s)indicate
7、sthatthereactionhasnotfailed(Fig.1A).Furthermore,becausemajordeletionsareusuallycontiguous,(4)resultsthatsuggestnoncontiguousdeletionsbasedontheabsenceofbandsusuallyreflectartifactualfailureofsomefragmentstoamplify.CompletePCRfailurecanbedistinguishedfromaninformativeno-amplificationresultbyaddi
8、ngacontrolampliconexternaltothetargetsequencetothereaction.(s'6)InadditiontomonitoringPCRfailureandartifacts,internalcontrolampliconscanbedesignedtoverifythepresenceoftargettemplate.Inmultiplexassayswherecloselyrelatedtem-pl
