文档介绍：oreplicationerrorsDefectionofMMRMSinstabilityLSSporadicMSILSMS错误产生机制Stutter又被称为影子带,或者DNA聚合酶滑脱产物。表现为PCR产物比主要扩增产物增加或减少一个或几个重复单位。MSIMAKERSINLSNationalCancerInstitute–sponsoredworkshopin19972mononucleotiderepeats(BAT25andBAT26)3dinucleotiderepeats(D2S123,D5S346,andD17S250)Revisedin2002 5mononucleotiderepeats(BAT25,BAT26,NR-21,NR-22,andNR-24)PromegaMSIAnalysisSystem5mononucleotiderepeats(BAT25,BAT26,MONO-27,NR-21andNR-24)2pentanucleotiderepeats(Penta-CandPenta-D)criteriaMSI-HReplicationerrors≥2makersMSI-L1MSS0实验流程1、基因DNA的提取2、多重荧光PCR3、毛细管电泳检测4、分析数据基因DNA的提取流程Promega试剂盒法分别挑选正常、肿瘤组织显微切割多重荧光PCRDiseaseMarkers20(2004)237–,’端作荧光标记JOE~GreenFL~BlueTMR~Black多重荧光PCRPCR反应体系(25μl):µlGoldST★-1µµlAmpliTaqGoldDNAPolymerase(5Units/µl)1-2ngDNAPCR条件:1cycle95◦Cfor11minutes;1cycle96◦Cfor1minute;10cycles94◦Cfor30sec-onds,ramp6secondsto56◦C,holdfor30seconds,ramp50secondsto70◦C,holdfor45seconds;20cyclesat90◦Cfor30seconds,ramp60secondsto56◦C,holdfor30seconds,ramp50secondsto70◦C,holdfor45seconds;60◦Cfor30minutesfinalextension;4◦(2004)237–,、分析微卫星位点经过荧光标记PCR扩增后,取适量PCR产物、分子内标(ILS600)溶于缓冲液(甲酰胺)中经遗传分析仪毛细管电泳进行扫描,通过检测片段长度来判断微卫星不稳定性。yper软件进行图像收集和分析,计算出微卫星等位变异扩增片段的大小。PromegaMSIAnalysisSystem,