Abstract Polymerase chain reaction (PCR) is the most commonly used method for nucleic acids amplification. PCR performance depends on several causes, among which the quality of primers is one of the main determinants affecting specificity, sensitivity and reliability of the reaction. Here, we report on the results of the detailed study devoted to the dimerization of the primers during PCR. The course and specificity of the reaction were studied on the model DNA templates as well as genomic DNA using primers that form amplifiable heterodimeric structures with different thermodynamic stability. It was confirmed that more than two 3′-overlapping nucleotides cause a considerable accumulation of primer dimers. It turned out that the presence of any DNA promotes the formation of dimers even for primers, which do not tend to nonspecific amplification in the absence of DNA. It was shown that dimerization could not be eliminated by commonly used techniques. Even the use of hot-start DNA polymerases does not prevent PD formation if primers with stable 3′-overlapping are employed. Despite several advantages of PCR with abutting primers, their close disposition has no benefits regarding the formation of PD if low-quality primers are utilized.

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PCR中引物质量对引物二聚体形成的影响

摘要 聚合酶链反应（PCR）是最常用的核酸扩增方法。PCR 性能取决于多种原因，其中引物的质量是影响反应特异性、灵敏度和可靠性的主要决定因素之一。在这里，我们报告了专门针对 PCR 过程中引物二聚化的详细研究结果。使用可形成具有不同热力学稳定性的可扩增异二聚体结构的引物，在模型 DNA 模板和基因组 DNA 上研究了反应过程和特异性。已证实超过两个 3'-重叠核苷酸会导致引物二聚体的大量积累。结果表明，即使是引物，任何 DNA 的存在都会促进二聚体的形成，在没有 DNA 的情况下，它们不会发生非特异性扩增。结果表明，常用技术不能消除二聚化。如果使用具有稳定 3'-重叠的引物，即使使用热启动 DNA 聚合酶也不能阻止 PD 的形成。尽管使用邻接引物进行 PCR 有几个优点，但如果使用低质量引物，它们的紧密布置对 PD 的形成没有任何好处。