Fusion primer driven racket PCR: A novel tool for genome walking

Jinfeng Pei; Tianyi Sun; Lingqin Wang; Zhenkang Pan; Xinyue Guo; Haixing Li

doi:10.3389/fgene.2022.969840

Fusion primer driven racket PCR: A novel tool for genome walking

Front Genet. 2022 Oct 18:13:969840. doi: 10.3389/fgene.2022.969840. eCollection 2022.

Authors

Jinfeng Pei^{1

2}, Tianyi Sun^{1

2

3}, Lingqin Wang^{1

2}, Zhenkang Pan^{1

2}, Xinyue Guo^{1

2}, 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 Key Laboratory of Poyang Lake Environment and Resource Utilization of Ministry of Education, School of Chemistry and Chemical Engineering, Nanchang University, Nanchang, China.

Abstract

The limitations of the current genome-walking strategies include strong background and cumbersome experimental processes. Herein, we report a genome-walking method, fusion primer-driven racket PCR (FPR-PCR), for the reliable retrieval of unknown flanking DNA sequences. Four sequence-specific primers (SSP1, SSP2, SSP3, and SSP4) were sequentially selected from known DNA (5'→3') to perform FPR-PCR. SSP3 is the fragment that mediates intra-strand annealing (FISA). The FISA fragment is attached to the 5' end of SSP1, generating a fusion primer. FPR-PCR comprises two rounds of amplification reactions. The single-fusion primary FPR-PCR begins with the selective synthesis of the target first strand, then allows the primer to partially anneal to some place(s) on the unknown region of this strand, producing the target second strand. Afterward, a new first strand is synthesized using the second strand as the template. The 3' end of this new first strand undergoes intra-strand annealing to the FISA site, followed by the formation of a racket-like DNA by a loop-back extension. This racket-like DNA is exponentially amplified in the secondary FPR-PCR performed using SSP2 and SSP4. We validated this FPR-PCR method by identifying the unknown flanks of Lactobacillus brevis CD0817 glutamic acid decarboxylase genes and the rice hygromycin gene.

Keywords: fusion primer; genome-walking; partial annealing; racket-like DNA; walking primer.