PCR引物特异性核查系统(PSC)的构建与应用

PSC:A system for PCR primer specificity checking and application

聚合酶链式反应(PCR)虽已广泛用于分子生物学研究中,然而PCR实验中的非特异性产物问题将直接影响PCR的效率,在多重PCR实验中更是如此。为了最大限度地降低非特异性产物的出现率,同时避免用户频繁使用Blast比对检查非特异性,我们开发了基于NCBI-Blast的引物评估和模板DNA特异性结合能力评估的核查系统PSC(Primer Specificity Checking,http://biocompute.bmi.ac.cn/PSC),并基于虚拟PCR实验确定了用于引物质量核查计算的多种参数,能够在线提供多个物种的引物特异性核查结果。该系统可以有效地对引物序列可能产生的所有非特异性扩增进行预测,有助于实验前引物优化或者对非特异扩增结果进行解释,最终达到提高PCR效率的目的。

Polymerase Chain Reaction （PCR） is widely used in biological research. The non- specific products obtained in PCR seri- ously interfere the efficiency of routine PCR experiments and especially multiplex PCR reactions because of cross hybridization. However, none efficient programs for primer specificity evaluation and are available till now. In order to avoid obtaining the non - specific am- plicons in PCR experiment, we built a web -based tool PSC （Primer Specificity Checking, http：//biocompute, bmi. ac. cn/PSC） to improve the accuracy of PCR by checking the match of the primers to DNA templates based on NCBI - Blast program. Databases of multiple species have been provided for primer specificity checking and also the optimal parameter of word size is suggested for evalua- tion after testing various of primer pairs based on different species. Focusing on predictiion of the possible non - specific amplicons, PSC can be used to optimize primer design or analyze the unexpected PCR results to improve the PCR efficiency.