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DNA polymerase preference determines PCR priming efficiency

机译：DNA聚合酶偏好决定PCR引发效率

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Background Polymerase chain reaction (PCR) is one of the most important developments in modern biotechnology. However, PCR is known to introduce biases, especially during multiplex reactions. Recent studies have implicated the DNA polymerase as the primary source of bias, particularly initiation of polymerization on the template strand. In our study, amplification from a synthetic library containing a 12 nucleotide random portion was used to provide an in-depth characterization of DNA polymerase priming bias. The synthetic library was amplified with three commercially available DNA polymerases using an anchored primer with a random 3’ hexamer end. After normalization, the next generation sequencing (NGS) results of the amplified libraries were directly compared to the unamplified synthetic library. Results Here, high throughput sequencing was used to systematically demonstrate and characterize DNA polymerase priming bias. We demonstrate that certain sequence motifs are preferred over others as primers where the six nucleotide sequences at the 3’ end of the primer, as well as the sequences four base pairs downstream of the priming site, may influence priming efficiencies. DNA polymerases in the same family from two different commercial vendors prefer similar motifs, while another commercially available enzyme from a different DNA polymerase family prefers different motifs. Furthermore, the preferred priming motifs are GC-rich. The DNA polymerase preference for certain sequence motifs was verified by amplification from single-primer templates. We incorporated the observed DNA polymerase preference into a primer-design program that guides the placement of the primer to an optimal location on the template. Conclusions DNA polymerase priming bias was characterized using a synthetic library amplification system and NGS. The characterization of DNA polymerase priming bias was then utilized to guide the primer-design process and demonstrate varying amplification efficiencies among three commercially available DNA polymerases. The results suggest that the interaction of the DNA polymerase with the primer:template junction during the initiation of DNA polymerization is very important in terms of overall amplification bias and has broader implications for both the primer design process and multiplex PCR.
机译：背景技术聚合酶链反应（PCR）是现代生物技术中最重要的发展之一。然而，已知PCR会引入偏差，尤其是在多重反应期间。最近的研究表明，DNA聚合酶是产生偏差的主要来源，特别是模板链上的聚合反应开始。在我们的研究中，使用包含12个核苷酸随机部分的合成文库进行扩增，以提供DNA聚合酶引发偏向的深入表征。使用带有随机3'六聚体末端的锚定引物，用三种市售DNA聚合酶扩增合成文库。标准化后，将扩增文库的下一代测序（NGS）结果直接与未扩增合成文库进行比较。结果在这里，高通量测序被用来系统地证明和表征DNA聚合酶引发的偏倚。我们证明，某些序列基序比其他序列基序更可作为引物，其中引物3'端的六个核苷酸序列以及引物位点下游的四个碱基对的序列可能会影响引物效率。来自两个不同商业供应商的同一家族中的DNA聚合酶偏爱相似的基序，而来自不同DNA聚合酶家族的另一种可商购获得的酶偏爱不同的基序。此外，优选的启动基序是富含GC的。通过从单引物模板扩增证实了某些序列基序的DNA聚合酶偏好性。我们将观察到的DNA聚合酶偏好性整合到引物设计程序中，该程序可指导将引物放置在模板上的最佳位置。结论使用合成文库扩增系统和NGS表征了DNA聚合酶引发的偏向。然后，利用DNA聚合酶引发偏倚的特征来指导引物设计过程，并证明三种市售DNA聚合酶之间变化的扩增效率。结果表明，DNA聚合酶与DNA聚合反应引发的引物：模板连接之间的相互作用就总体扩增偏差而言非常重要，并且对引物设计过程和多重PCR都具有更广泛的意义。

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