定量PCR的非同源对照模板的构建  

Generation of Non-homologous Competitor DNA for Competitive Quantitative PCR

作　　者：陈燃[1] 金詟[1] 毛裕民[1] 

机构地区：[1]复旦大学生命科学学院遗传所遗传工程国家重点实验室,上海200433

出　　处：《遗传》2001年第3期247-250,共4页Hereditas（Beijing)

基　　金：国家自然科学基金!资助项目 ( 3 970 0 0 82 )

摘　　要：建立了HCVRNA和hTERTmRNA的竞争定量PCR系统。对照模板的构建方法是 :利用计算机辅助优选设计连接引物 ,低严紧型扩增大肠杆菌DNA ,回收并克隆预期的DNA片段。该片段与靶序列除两端引物序列完全相同外 ,无同源性 ,因此可作为非同源对照模板。这种构建非同源对照模板的方法 ,简便易行 。It cannot be neglected sometimes that the error caused by the “heteroduplex DNA” that occurs accompanying with the homologous competitor DNA which is usually used in the competitive quantitative PCR (CQ-PCR). Here a method is developed to generate non-homologous competitor DNA for CQ-PCR detection of the interest DNA, based on low-stringency amplification of cross-species' DNA with a pair of linker-primers which are designed according to partly homologous sites of the interest DNA primers in cross-species' DNA. With the method, the non-homologous competitor DNAs for the HCV RNA and hTERT mRNA are generated from E. coli DNA respectively, then the CQ-PCR systems are established for the 2 species' RNAs with the RRTR (Repeated reverse transcription reaction). The method is multi-adaptive and easy to apply.

关 键 词：竞争定量PCR 对照模板 异源双链 大肠杆菌 

分 类 号：Q933[生物学—微生物学] Q75