Deconstructing the Polymerase Chain Reaction: Understanding and Correcting Bias Associated with Primer Degeneracies and Primer-Template Mismatches

The polymerase chain reaction (PCR) is sensitive to mismatches between primer and template, and mismatches can lead to inefficient amplification of targeted regions of DNA template. In PCRs in which a degenerate primer pool is employed, each primer can behave differently. Therefore, inefficiencies due to different primer melting temperatures within a degenerate primer pool, in addition to mismatches between primer binding sites and primers, can lead to a distortion of the true relative abundance of targets in the original DNA pool. A theoretical analysis indicated that a combination of primer-template and primer-amplicon interactions during PCR cycles 3–12 is potentially responsible for this distortion. To test this hypothesis, we developed a novel amplification strategy, entitled “Polymerase-exonuclease (PEX) PCR”, in which primer-template interactions and primer-amplicon interactions are separated. The PEX PCR method substantially and significantly improved the evenness of recovery of sequences from a mock community of known composition, and allowed for amplification of templates with introduced mismatches near the 3’ end of the primer annealing sites. When the PEX PCR method was applied to genomic DNA extracted from complex environmental samples, a significant shift in the observed microbial community was detected. Furthermore, the PEX PCR method provides a mechanism to identify which primers in a primer pool are annealing to target gDNA. Primer utilization patterns revealed that at high annealing temperatures in the PEX PCR method, perfect match annealing predominates, while at lower annealing temperatures, primers with up to four mismatches with templates can contribute substantially to amplification. The PEX PCR method is simple to perform, is limited to PCR mixes and a single exonuclease step which can be performed without reaction cleanup, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible.

聚合酶链式反应（polymerase chain reaction，PCR）对引物（primer）与模板（template）间的错配十分敏感，错配会导致DNA模板靶区域的扩增效率降低。当使用简并引物库（degenerate primer pool）进行PCR时，不同引物的行为存在差异。因此，除引物结合位点与引物间的错配外，简并引物库内不同引物的解链温度（melting temperature）差异引发的扩增低效问题，可能会扭曲原始DNA库中靶标真实的相对丰度（relative abundance）。一项理论分析（theoretical analysis）表明，PCR循环3至12阶段中，引物-模板相互作用（primer-template interaction）与引物-扩增子（primer-amplicon interaction）的共同作用是导致该丰度扭曲现象的潜在原因。为验证该假说（hypothesis），我们开发了一种名为“聚合酶-核酸外切酶（Polymerase-exonuclease，PEX）PCR”的新型扩增策略，该策略将引物-模板相互作用与引物-扩增子相互作用分离开来。PEX PCR方法显著提升了已知组成模拟群落（mock community）的序列回收均匀度，且可对引物结合位点3’端存在引入错配的模板进行扩增。将PEX PCR方法应用于复杂环境样品（complex environmental samples）提取的基因组DNA（genomic DNA）时，检测到观测到的微生物群落（microbial community）发生了显著变化。此外，PEX PCR方法还提供了一种可识别引物库中哪些引物能与靶基因组DNA结合的机制。引物利用模式（primer utilization pattern）显示，在PEX PCR的高退火温度条件下，完全匹配退火（perfect match annealing）占据主导；而在较低退火温度下，与模板存在至多4个错配的引物也可显著参与扩增。PEX PCR方法操作简便，仅需常规PCR反应体系与单次核酸外切酶（exonuclease）处理步骤，且无需进行反应产物纯化即可完成，适用于使用简并引物库或存在引物-模板错配风险的扩增反应。