MSI检测技术.ppt

文档介绍：MSI检测技术微卫星简要介绍现用微卫星检测技术流程多重荧光PCR技术概略介绍毛细管电泳概略介绍微卫星检测的临床检测可行性MSI检测技术摘要MSSimplerepeatsequencesof1to6basepairs单核苷酸型:AAAAAAAA..双核苷酸型:ATATATAT..三核苷酸型:ATCATCATC..…….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–,