Single-primer PCR correction: a strategy for false-positive exclusion
Single-primer PCR correction: a strategy for false-positive exclusion
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单引物 PCR 校正：假阳性排除策略
DOI：
10.4238/vol10-1gmr988
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发表时间：
2011-01-01
影响因子：
0.4
通讯作者：
Guan, S. C.
中科院分区：
文献类型：
--
作者：
Ma, J.;Wang, P. W.;Guan, S. C.
聚合酶链式反应（PCR）技术在分子生物学研究中发挥着重要作用，但假阳性和非特异性PCR扩增一直困扰着许多研究人员。目前，PCR体系优化的研究主要集中在基于双引物的PCR产物上。本研究表明，在基于PCR的目的基因克隆、转基因植物检测、简单序列重复标记辅助选择和mRNA差异展示中，当PCR条件受到高度限制时，基于单引物与DNA模板结合的PCR扩增是获得假阳性结果、片段杂质和非特异性片段扩增的重要因素。在这里，我们比较了单引物和双引物扩增，并提出了“单引物PCR校正”； PCR技术的改进消除了基于单引物的非特异性PCR扩增引起的干扰，并提高了实验的精确度和成功率。虽然对于某些类型的实验，单引物PCR校正的改善效果存在差异，但在我们的实验中，通过这种方式可以将精确度和成功率提高12-50%。
Polymerase chain reaction (PCR) technology plays an important role in molecular biology research, but false-positive and nonspecific PCR amplification have plagued many researchers. Currently, research on the optimization of the PCR system focuses on double-primer-based PCR products. This research has shown that PCR amplification based on single-primer binding to the DNA template is an important contributing factor to obtaining false-positive results, fragment impurity, and nonspecific fragment amplification, when the PCR conditions are highly restricted during PCR-based target gene cloning, detection of transgenic plants, simple-sequence repeat marker-assisted selection, and mRNA differential display. Here, we compared single-and double-primer amplification and proposed "single-primer PCR correction"; improvements in PCR that eliminate interference caused by single-primer-based nonspecific PCR amplification were demonstrated and the precision and success rates of experiments were increased. Although for some kinds of experiments, the improvement effect of single-primer PCR correction was variable, the precision and success rate could be elevated at 12-50% in our experiment by this way.