Fork PCR: a universal and efficient genome-walking tool

Hao Pan; Xinyue Guo; Zhenkang Pan; Rongrong Wang; Bingkun Tian; Haixing Li

doi:10.3389/fmicb.2023.1265580

Fork PCR: a universal and efficient genome-walking tool

Front Microbiol. 2023 Sep 22:14:1265580. doi: 10.3389/fmicb.2023.1265580. eCollection 2023.

Authors

Hao Pan^{1

2

3}, Xinyue Guo^{2

3}, Zhenkang Pan^{2

3}, Rongrong Wang^{2

3}, Bingkun Tian^{2

3}, Haixing Li^{2

3}

Affiliations

¹ School of Chemistry and Chemical Engineering, Nanchang University, Nanchang, China.
² State Key Laboratory of Food Science and Resources, Nanchang University, Nanchang, China.
³ Sino-German Joint Research Institute, Nanchang University, Nanchang, China.

Abstract

The reported genome-walking methods still suffer from some deficiencies, such as cumbersome experimental steps, short target amplicon, or deep background. Here, a simple and practical fork PCR was proposed for genome-walking. The fork PCR employs a fork primer set of three random oligomers to implement walking task. In primary fork PCR, the low-stringency amplification cycle mediates the random binding of primary fork primer to some places on genome, producing a batch of single-stranded DNAs. In the subsequent high-stringency amplification, the target single-strand is processed into double-strand by the site-specific primer, but a non-target single-stranded DNA cannot be processed by any primer. As a result, only the target DNA can be exponentially amplified in the remaining high-stringency cycles. Secondary/tertiary nested fork PCR(s) further magnifies the amplification difference between the both DNAs by selectively enriching target DNA. The applicability of fork PCR was validated by walking several gene loci. The fork PCR could be a perspective substitution for the existing genome-walking schemes.

Keywords: fork primer set; genome-walking; nested PCR; primer overlap; randomly partial annealing.

Grants and funding

The authors declare financial support was received for the research, authorship, and/or publication of this article. This study was funded by the Jiangxi Provincial Department of Science and Technology, China (Grant Number 20225BCJ22023) and the National Natural Science Foundation of China (Grant Number 32160014).