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
MS
Simple repeat sequences of 1 to 6 base pairs
单核苷酸型:AAAAAAAA..
双核苷酸型:ATATATAT..
三核苷酸型:ATCATCATC..
…….
MS
Prone to replication errors
Defection of MMR
MS instability
LS
Sporadic MSI
LS
实验流程
1、基因DNA的提取
2、多重荧光PCR
3、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PCR
Disease Markers 20 (2004) 237–250
Jeffery W. Bacher, Laura A. Flanagan
上游引物的5’端作荧光标记
JOE~Green FL ~Blue TMR~Black
多重荧光PCR
PCR反应体系(25μl):
µl GoldST★R 10X Buffer
-1 µM each primers
µl AmpliTaq Gold DNA Polymerase(5Units/ µl)
1-2ng DNA
PCR条件:1 cycle 95◦C for 11minutes;
1 cycle 96◦C for 1 minute;
10 cycles 94◦C for 30 sec-onds, ramp 6 seconds to 56◦C, hold for 30 seconds,ramp 50 seconds to 70◦C, hold for 45 seconds;
20 cycles at 90◦C for 30 seconds, ramp 60 seconds to 56◦C,hold for 30 seconds, ramp 50 seconds to 70◦C, hold for45 seconds;
60◦C for 30 minutes final extension;
4◦C hold.
Disease Markers 20 (2004) 237–250
Jeffery W. Bacher, Laura A. Flanagan