pyrosequencing焦磷酸测序.ppt

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1、焦磷酸测序法的原理及应用（Pyrosequencing遗传分析技术）pyro=pyrophosphate焦磷酸一种全新的基于酶级联反应的新型遗传分析技术;一套完整的实验方案；诸多领域的广泛应用……Pyrosequencing遗传分析技术Pyrosequencing这项专利技术完全由Biotage公司拥有PPiATPPrincipleofPyrosequencingAPS＋Luciferin＋硫酸化酶双磷酸酶荧光素酶oxyluciferinDetectionofthelightlighttimeCGTTCCACCT每次向反应体系中加入一种dNTP相应位置的峰代表该种dNTP的掺入情

2、况峰高与掺入的核苷酸数量成正比多余的dNTP在加入下一种dNTP前就被降解AcompletesolutionforaccurategeneticdeterminationPyrosequencing系统平台ReagentKitsSamplePrepWorkstationSoftwareInstrumentationPyroMarkTMIDCompletesolutionforClinicalMicrobiologyPyrosequencing系统平台AssayDesignSWSamplePrepPyrosequencingFeaturingPyroGoldchemistryIde

3、ntiFireTMSWPyrosequencing技术的流程PCR试样预处理，获得单链模板Pyrosequencing序列分析15min/96samplesSQA:35min/96samplesSNP:10min/96samples1-2h/96samples结果分析实验设计PeakheightisproportionaltothenumberofincorporatednucleotidesAllele1:ACTGAllele2:GCTG杂合子标本:Genotypesareclearlydistinguished纯合子杂合子ACTGCCTGCTGCCTA/GCTGCCTIns

4、ertions/deletionsSinglenucleotideInDels(e.g.[C])MultiplenucleotideInDels(e.g.[CGACGGT])Thesoftwaresupportsanalysisofinsertions/deletions(InDels)CYP2D6-A2637del(Allele3)Sequence(reverse):TCC[T]GTGT/TT/--/-Largedeletion3’ATGTGGCATTCCAA5’...ACGTACGCTTACACCGTAAGGTTCTAAAGGTG[CACCATGACTGGGGTTA]CAGT

5、CATC...Microsatellites:TG[AATG]nTTTGGGCAAAT8/128/8478478(9)(10)(11)(12)Tri-andtetra-allelicSNPsC/CT/TG/GC/TC/GT/GGenotypingoftri-allelicSNP(C/T/G)Multiplepolymorphisms3’TAAGCCGAATG5’…AACATTCGGCTTACAT/GGAATGAA/C/G/TCGGTACGAACGATTTTAGA...MultiplexingSNPslocatedondifferentfragments…...…..oronthe

6、samePrincipleofmultiplexingDesignofsequencingprimersanddispensationorder3,5refref00,511,522,53RelativelightunitsTAGCGCACGATGT[A/G]CCTGCCACC[A/G]TTGGGCombinedTGGACCACC[A/G]TGGTTGAG[A/G]CTGCCTG12TTGAG[A/G]CTGCCTG00,511,52TAGCGCACGATGTTGGACCACC[A/G]TGG00,511,52TAGCTGCACGATGUsercreatesaSNP/mutati

7、onsequencedatabaseSoftwarecalculatestheoreticalgenotypingresultsEachDNAfragmentisassignedacolorQuantitativepeakheightsRelativepeakheight==peak1heightpeak1height+peak2heightAllelefrequency==relativepeakheightx100(%)RelativepeakheightAllelefreq