Mutation Detection by Real-Time PCR A Simple, Robust and Highly Selective Method 英文参考文献.docVIP

MutationDetectionbyReal-TimePCR:ASimple,Robust andHighlySelectiveMethod JohnMorlan,JoffreBaker,DominickSinicropi* GenomicHealth,Inc.,RedwoodCity,California,UnitedStatesofAmerica Abstract Background:Moleculartestsfordiagnosisofdisease,particularlycancer,aregainingincreasedacceptancebyphysicians andtheirpatientsfordiseaseprognosisandselectionoftreatmentoptions.Geneexpressionprofilesandgeneticmutations arekeyparametersusedforthemolecularcharacterizationoftumors.Avarietyofmethodsexistformutationanalysisbut thedevelopmentofassayswithhighselectivitytendstorequireaprocessoftrialanderror,andfewarecompatiblewith real-timePCR.Wesoughttodevelopareal-timePCR-basedmutationassaymethodologythatsuccessfullyaddressesthese issues. Methodology/PrincipalFindings:ThemethodwedescribeisbasedonthewidelyusedTaqManHreal-timePCRtechnology, andcombinesAllele-SpecificPCRwithaBlockingreagent(ASB-PCR)tosuppressamplificationofthewildypeallele.ASB-PCR can be used for detection of germ line or somatic mutations in either DNA or RNA extracted from any type of tissue, including formalin-fixed paraffin-embedded tumor specimens. A set of reagent design rules was developed enabling sensitive andselective detection of single pointsubstitutions, insertions,or deletions against abackground ofwild-type alleleinthousand-foldorgreaterexcess. Conclusions/Significance: ASB-PCR is a simple and robust method for assaying single nucleotide mutations and polymorphisms within the widely used TaqManH protocol for real time RT-PCR. The ASB-PCR design rules consistently producehighlyselectivemutationassayswhileobviatingtheneedforredesignandoptimizationoftheassayreagents.The methodiscompatiblewithformalin-fixedtissueandsimultaneousanalysisofgeneexpressionbyRT-PCRonthesameplate. No proprietary reagents other than those for TaqMan chemistry are required, so the method can be performed in any researchlaboratorywithreal-timePCRcapability. Citation: Morlan J, Baker J, Sinicropi D (2009) Mutation Detection by Real-Time PCR: