定量PCR的非同源对照模板的构建

Generation of Non-homologous Competitor DNA for Competitive Quantitative PCR

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.