DNA polymerase preference determines PCR priming efficiency

Wenjing Pan,Robert J Zahorchak,Miranda Byrne-Steele,Jian Han,Chunlin Wang,Scott Clemmons,Stanley Lu

doi:10.1186/1472-6750-14-10

Abstract

BackgroundPolymerase 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.ResultsHere, 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.ConclusionsDNA 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.

Highlights

Polymerase chain reaction (PCR) is one of the most important developments in modern biotechnology
In order to define this source of DNA polymerase bias, we carefully examined the contribution of the DNA sequence from a ten base pair window surrounding the primer:template junction including six base pairs of the primer:template duplex, which rests in the palm of the polymerase prior to nucleotide addition [7,8], and the four bps of singlestranded DNA template immediately following the 90° kink at the junction, which we termed the “runway”
Amplification experiments and barcode analysis In order to define the DNA polymerase bias, we created a synthetic library (SL) of sequences that are identical in sequence with the exception of a 12 nucleotide random insertion (Figure 3A)

Summary

Introduction

Polymerase chain reaction (PCR) is one of the most important developments in modern biotechnology. Recent studies have implicated the DNA polymerase as the primary source of bias, initiation of polymerization on the template strand. PCR is known to introduce biases during amplification, during multiplex PCR when several templates are amplified simultaneously [1,2]. Recent evaluations of biases generated in high throughput sequencing data have pinpointed the amplification step as the primary cause [4,5]. Factors such as thermocycler make, model, and ramping speed were demonstrated to affect the uniformity of the amplified library [4]. The DNA polymerase was identified as the primary source of bias with a variety of commercially available DNA polymerases skewing the amplification profile of the Neandertal genome with regard to GC content and template length [5]

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