Wristwatch PCR: A Versatile and Efficient Genome Walking Strategy

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.

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

“…In some situations, more than one clear target DNA band appeared (Figure 1). This multiple-band phenomenon is common in randomly primed PCRs and occurs due to the annealing of the walking primers to multiple sites on the unknown flank in the low-stringency cycle (Chang et al, 2018;Wang et al, 2022). At most two major bands appeared in each secondary FPR-PCR in the present study, much fewer than the approximately five bands reported in the other methods (Chang et al, 2018;Wang et al, 2022).…”

“…Additionally, the ability to perform parallel simultaneous experiments provides a further guarantee for the success of FPR-PCR, as at least one FPR-PCR will give a positive outcome. FPR-PCR provides a high success rate similar to those reported for randomly primed PCRs, such as partially overlapping primer-based PCR ( Li et al, 2015 ), stepwise partially overlapping primer-based PCR ( Chang et al, 2018 ), and wristwatch PCR ( Wang et al, 2022 ).…”

“…The currently available PCR-based genome-walking methods are unsatisfactory due to limitations including low success rates, high background, and complicated procedures ( Myrick and Gelbart, 2002 ; Wang et al, 2022 ). Therefore, practical PCR-based genome-walking methods have been rigorously pursued.…”

“…The primary amplification in existing randomly primed PCR methods requires a walking primer with a random sequence, in addition to the outermost SSP. Therefore, three types of undesired species are generally formed, including (I) SSP alone; (II) the combination of SSP and walking primer; and (III) the walking primer alone ( Liu and Whittier, 1995 ; Kotik, 2009 ; Wang et al, 2022 ). In the FPR-PCR primary reaction, however, the fusion primer acts as not only SSP but also the walking primer.…”

“…This multiple-band phenomenon is common in randomly primed PCRs and occurs due to the annealing of the walking primers to multiple sites on the unknown flank in the low-stringency cycle ( Chang et al, 2018 ; Wang et al, 2022 ). At most two major bands appeared in each secondary FPR-PCR in the present study, much fewer than the approximately five bands reported in the other methods ( Chang et al, 2018 ; Wang et al, 2022 ). As mentioned, the amplicons resulting from the primary FPR-PCR encompassed the fusion primer and its inverted repeat.…”