摘要
目的建立一种可消除逆转录聚合酶链反应(reverse transcriptase-polymerase chain reaction,RT-PCR)中非特异条带生成,提高逆转录聚合酶链反应精确度的PCR体系。方法最近作者报道了硫化修饰的引物结合具有校正活性聚合酶可提高PCR反应特异性,但其抑制非特异性扩增的效率有待进一步验证和精确测定。用小鼠窖蛋白1(caveolin-1,Cav1)中1对引物,ECV304细胞、HepG2细胞、小鼠肝、主动脉血管组织逆转录cDNA以及含人窖蛋白基因的倍比稀释质粒为模板,比较普通RT-PCR反应与硫化修饰引物结合不同校正活性聚合酶对非特异性扩增的抑制效率。结果引物3′末端硫化修饰结合具有校正活性的聚合酶可特异性关闭PCR反应中引物3′末端与模板非特异结合而引起的非特异性扩增,只允许引物3′末端与模板完全匹配的延伸反应得以进行。与普通PCR反应相比,能有效抑制50μL体系中,50ng约含2×104不匹配模板拷贝数的非特异扩增。结论引物3′端引入抗酶切的硫化修饰后,引物与模板非特异结合将由于校正活性聚合酶长期停留在试图纠正引物与模板不一致状态而得不到有效延伸,提高了PCR反应的特异性。
Objective To construct a reverse transcriptase-polymerase chain reaction （RT-PCR） approach that can improve the specificity of primers while dropping down the nonspecifie amplification. Methods In the recent study we reported a new RT-PCR assay which improved markedly the specificity. However its efficiency of regressing nonspecif. ic amplification remains to be accurately checked and further documented. In primer design, we looked over again some sequences that showed differences at 5＇ or 3＇ ends between human CAV1 and mouse Cav1 genes, cDNAs and the diluted plasmids which harbored the sequence of human CAV1 or mouse Cav1 gene were chosen as the templates. The ordinary PCR compared with one, of which primers modified by phesphorothioate and combined with proofreading polymerase, for their effieieneies of nonspeeifie amplification inhibited. Results Taq DNA polymerase without proofreading activity could efficiently catalyze the extension of primers with a single or multiple mismatched base pairs at the 3＇ temainns, but the kind of primer extension can be effectively blocked by phesphorothioate modified primers combined with proofreading polymerase. Compared with ordinary PCR reaction, this new PCR method can effectively regress the primer mismatched amplification of 50 ng DNA almost equaling to 2×10^4 unmatched template copies in a final volume of 50μL. Conclusion Compared with the first generation of polymerases with or without proofreading activities mediating RT-PCR reaction, the introduction of nuclease-resistant 3＇modifed primers （3＇ phesphorothioate primer extension） can offer more simplicity, accuracy, and also decrease cost.
出处
《中华医学遗传学杂志》
CAS
CSCD
北大核心
2006年第1期59-62,共4页
Chinese Journal of Medical Genetics
基金
国家自然科学基金(30400265)
湖南省自然基金(03JJY3037
03JJY5009)~~
