Wristwatch PCR: A Versatile and Efficient Genome Walking Strategy

Lingqin Wang; Mengya Jia; Zhaoqin Li; Xiaohua Liu; Tianyi Sun; Jinfeng Pei; Cheng Wei; Zhiyu Lin; Haixing Li

doi:10.3389/fbioe.2022.792848

Wristwatch PCR: A Versatile and Efficient Genome Walking Strategy

Front Bioeng Biotechnol. 2022 Apr 12:10:792848. doi: 10.3389/fbioe.2022.792848. eCollection 2022.

Authors

Lingqin Wang^{1

2}, Mengya Jia^{1

2}, Zhaoqin Li³, Xiaohua Liu^{1

2}, Tianyi Sun^{1

2

4}, Jinfeng Pei^{1

2}, Cheng Wei^{1

2}, Zhiyu Lin^{1

2

4}, Haixing Li^{1

2}

Affiliations

¹ State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, China.
² Sino-German Joint Research Institute, Nanchang University, Nanchang, China.
³ Charles W. Davidson College of Engineering, San Jose State University, San Jose, CA, United States.
⁴ Key Laboratory of Poyang Lake Environment and Resource Utilization, Ministry of Education, School of Environmental and Chemical Engineering, Nanchang University, Nanchang, China.

Abstract

Genome walking is a method used to retrieve unknown flanking DNA. Here, we reported wristwatch (WW) PCR, an efficient genome walking technique mediated by WW primers (WWPs). WWPs feature 5'- and 3'-overlap and a heterologous interval. Therefore, a wristwatch-like structure can be formed between WWPs under relatively low temperatures. Each WW-PCR set is composed of three nested (primary, secondary, and tertiary) PCRs individually performed by three WWPs. The WWP is arbitrarily annealed somewhere on the genome in the one low-stringency cycle of the primary PCR, or directionally to the previous WWP site in one reduced-stringency cycle of the secondary/tertiary PCR, producing a pool of single-stranded DNAs (ssDNAs). A target ssDNA incorporates a gene-specific primer (GSP) complementary at the 3'-end and the WWP at the 5'-end and thus can be exponentially amplified in the next high-stringency cycles. Nevertheless, a non-target ssDNA cannot be amplified as it lacks a perfect binding site for any primers. The practicability of the WW-PCR was validated by successfully accessing unknown regions flanking Lactobacillus brevis CD0817 glutamate decarboxylase gene and the hygromycin gene of rice. The WW-PCR is an attractive alternative to the existing genome walking techniques.

Keywords: genome walking; partially annealing; wristwatch PCR; wristwatch primer; wristwatch-like DNA.