DNA polymerase preference determines PCR priming efficiency.
DNA polymerase preference determines PCR priming efficiency.
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DOI：
10.1186/1472-6750-14-10
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发表时间：
2014-01-30
影响因子：
3.5
通讯作者：
Han J
中科院分区：
文献类型：
--
作者：
Pan W;Byrne-Steele M;Wang C;Lu S;Clemmons S;Zahorchak RJ;Han J
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聚合酶是偏差的主要来源，特别是在模板链上引发聚合。在我们的研究中，从含有12个核苷酸的随机部分的合成文库中扩增，用于提供DNA聚合酶引发偏倚的深入表征。使用具有随机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）结果与未扩增的合成文库直接比较。在这里，高通量测序用于系统地证明和表征DNA聚合酶引发偏倚。我们证明了某些序列基序优于其他序列基序作为引物，其中引物3'端的6个核苷酸序列以及引发位点下游4个碱基对的序列可能影响引发效率。来自两个不同商业供应商的同一家族中的DNA聚合酶偏好相似的基序，而来自不同DNA聚合酶家族的另一种市售酶偏好不同的基序。此外，优选的引发基序是富含GC的。通过从单引物模板扩增来验证DNA聚合酶对某些序列基序的偏好。我们将观察到的DNA聚合酶的偏好纳入引物设计程序中，该程序指导引物在模板上的最佳位置。使用合成文库扩增系统和NGS表征DNA聚合酶引发偏倚。然后利用DNA聚合酶引发偏差的表征来指导引物设计过程，并证明三种市售DNA聚合酶之间不同的扩增效率。结果表明，DNA聚合酶与引物的相互作用：模板连接在DNA聚合的起始过程中是非常重要的整体扩增的偏见方面，引物设计过程和多重PCR具有更广泛的影响。
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. 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. 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.
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