Fluorescent Duplex Allele-Specific PCR and Amplicon Melting for Rapid Homogeneous mtDNA Haplogroup H Screening and Sensitive Mixture Detection 英文参考文献.docVIP

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Fluorescent Duplex Allele-Specific PCR and Amplicon Melting for Rapid Homogeneous mtDNA Haplogroup H Screening and Sensitive Mixture Detection 英文参考文献.doc

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Fluorescent Duplex Allele-Specific PCR and Amplicon Melting for Rapid Homogeneous mtDNA Haplogroup H Screening and Sensitive Mixture Detection 英文参考文献

FluorescentDuplexAllele-SpecificPCRandAmplicon MeltingforRapidHomogeneousmtDNAHaplogroupH ScreeningandSensitiveMixtureDetection HaraldNiedersta¨tter*,WaltherParson InstituteofLegalMedicine,InnsbruckMedicalUniversity,Innsbruck,Austria Abstract Background:ForlargescalestudiesaimingatabetterunderstandingofmitochondrialDNA(mtDNA),sequencevariationin particularmthaplogroups(hgs)andpopulationstructure,reliablelow-costhigh-throughputgenotypingassaysareneeded. Furthermore, methods facilitating sensitive mixture detection and relative quantification of allele proportions are indispensable for the study of heteroplasmy, mitochondrial sequence evolution, and mitochondrial disorders. Here the properties of a homogeneous competitive duplex allele specific PCR (ARMS) assay were scrutinized in the light of these requirements. Methodology/Principal Findings: A duplex ARMS assay amplifying either the ancestral mtDNA 2706G allele (non-hg H samples)orthederived7028Callele(hgHsamples)inthepresenceofSYBRGreenfluorescentreporterdyewasdeveloped andcharacterized.Productdetection,allelecalling,andhginferencewerebasedontheamplicon-characteristicmelting- pointtemperaturesobtainedwithon-linepost-PCRfluorescentdissociationcurveanalysis(DCA).Theanalyticalwindowof theassaycoveredatleast5ordersofmagnitudeoftemplateDNAinputwithadetectionlimitinthelowpicogramrangeof genomicDNA.Asetofforensicallyrelevanttestspecimenswasanalyzedsuccessfully.ThepresenceofmtDNAmixtureswas detectedoverabroadrangeofinputDNAamountsandmixtureratios,andtheestimationofalleleproportionsinsamples withknowntotalmtDNAcontentwasfeasiblewithlimitations.AqualifiedDNAanalystsuccessfullyanalyzed,2,200DNA extractswithinthreeregularworkingdays,withoutusingroboticlab-equipment.Byperformingtheamplificationon-line, theassayalsofacilitatedabsolutemtDNAquantification. Conclusions:Althoughthisassaywasdevelopedjustforaparticularpurpose,theapproachisgeneralinthatitispotentially suitableinabroadvarietyofassay-layoutsformanyotherapplications,inc