Stepwise partially overlapping primer-based PCR for genome walking

Kunpeng Chang,Shuixing Wang,Hongjing Wu,Lijuan Nie,Xiaofei Shi,Haixing Li,Qiong Wang

doi:10.1186/s13568-018-0610-7

Kunpeng Chang, Shuixing Wang + Show 5 more

Open Access

https://doi.org/10.1186/s13568-018-0610-7

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Abstract

A stepwise partially overlapping primer-based PCR (SWPOP-PCR) method for isolating flanking unknown DNA regions was developed, which comprises three rounds of nested PCRs sequentially driven by SWPOP primer-nested specific primer pairs. SWPOP primer set is characterized by a partial overlap of 10 bp with 3′-part of the latter primer is identical to 5′-part of the former one, which makes the SWPOP primer in use anneal to SWPOP site of the prior PCR product only at relatively low temperature. For each PCR, target single-stranded DNA primed by the SWPOP primer in the exclusive one low-stringency cycle is converted into double-stranded form in the following high-stringency cycle due to the presence of a perfect annealing site for the specific primer. This double-stranded DNA bounded by the specific primer and the SWPOP primer is exponentially amplified in the remaining high-stringency cycles. Non-target single-stranded DNA, however, cannot be amplified given the lack of perfect complementary sequences for any primers. Therefore, the partial overlap of a SWPOP primer set preferentially synthesizes target products but inhibits nonspecific amplification. We successfully exploited SWPOP-PCR to obtain the DNA sequences flanking glutamate decarboxylase gene (gadA) locus in Lactobacillus brevis NCL912 and hygromycin gene (hyg) integrated in rice.

Highlights

Numerous PCR-based genome walking methodologies have been developed for identification and isolation of neighboring unknown DNA sequences adjacent to known genomic regions, which can be classified into three main categories (Kotik 2009; Leoni et al 2011): (I) inverse PCR (Ochman et al 1988); (II) ligation mediated PCR (Mueller and Wold 1989; Arnold and Hodgson 1991; Jones and Winistorfer 1992; Yan et al 2003; Ji and Braam 2010); and (III) randomly primed PCR (Liu and Whittier 1995; Tan et al 2005; Wang et al 2013)
A gene-specific primer set consists of three nested primers [SP-P, SP-S, specific primer for primary (SP-T)], which were designed based on the DNA sequences of glutamate decarboxylase gene locus (GenBank accession number JX074764) of L. brevis Lactobacillus brevis NCL912 (NCL912) (Li et al 2013) and hygromycin gene (KF206149.1) integrated in the genome of rice, respectively
In our PCR method, a SWPOP-P primer should find an adapted site in the plate given the fact that one super low-stringency (25 °C) cycle was performed in primary PCR

Summary

Introduction

Numerous PCR-based genome walking methodologies have been developed for identification and isolation of neighboring unknown DNA sequences adjacent to known genomic regions, which can be classified into three main categories (Kotik 2009; Leoni et al 2011): (I) inverse PCR (Ochman et al 1988); (II) ligation mediated PCR (Mueller and Wold 1989; Arnold and Hodgson 1991; Jones and Winistorfer 1992; Yan et al 2003; Ji and Braam 2010); and (III) randomly primed PCR (Liu and Whittier 1995; Tan et al 2005; Wang et al 2013). The first two categories rely on labor-intensive and time-consuming restriction digestion and ligation of genomic DNA before PCR amplification (Rosenthal and Jones 1990; Acevedo et al 2008; Leoni et al 2008, 2010; Trinh et al 2012b; Spalinskas et al 2013). We developed a partially overlapping primerbased PCR (POP-PCR) method for genome walking, which employed a set of POP primers having identical 3′ ends of 10 bp to suppress the amplification of non-target products while effectively enrich the target molecules. We present a novel genome walking strategy, termed stepwise partially overlapping primerbased PCR (SWPOP-PCR), which is easier to operate and more economical than the conventional POP-PCR. The feasibility of the new method was tested by retrieving

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Conclusion