Background

Genome walking (GW) is a molecular tool for cloning unknown regions flanking known DNAs, facilitating methods such as gene cloning, identifying DNA mutations, and analyzing transgenic sites [2–5]. Up to date, three types of GW were available: random PCRs, genome library-based techniques, and restriction-ligation-based PCRs. Among them, genome library-based techniques are time-consuming and inefficient, due to requiring the construction and screening of a genomic DNA library, and restriction-ligation-based PCRs require the digestion of genomic DNA and the subsequent ligation of the digested product prior to amplification. Comparatively, random PCRs are faster and more efficient, as the extra steps prior to amplification are omitted [6–10].

Random PCRs generally require two to three rounds of nested amplification. In primary amplification, a low-temperature annealing cycle allows the walking primer (WP) to randomly anneal to the unknown flank, thereby synthesizing a target DNA comprising a known region and its unknown flank. The following round or two of nested amplification further enrich this DNA, ultimately achieving the so-called genome walking [11–15]. However, due to the use of WP and at least one low-temperature cycle in each round of amplification, three types of non-target amplicons will be produced. Type I is synthesized by GSP alone; type II is synthesized by GSP and WP; and type III is synthesized by WP alone. Types I and II can be easily removed in the next PCR because they lack an authentic binding site for the sequence-specific primer (SSP). The real challenge is how to eliminate the type III non-target product [16–19]. Existing random PCRs, such as thermal asymmetric interlaced PCR [20], fusion primer and nested integrated PCR [21], and partially overlapping primer-based PCR [22,23], have their reliability compromised due to the accumulation of this non-target product. Therefore, a truly reliable genome-walking scheme should be able to fundamentally overcome this non-target amplification, which has always been pursued by researchers [24–27].

In this study, a bridging PCR-based genome-walking protocol was designed. The main innovation of this PCR is the use of a bridging primer (BP) in secondary PCR, which is made by attaching an oligomer (or tail primer, TP) to the 5′ end of the WP 5′ region. As a result, in secondary PCR, the primary non-target product defined by the WP—namely, the main contributor to background—is lengthened by the BP at both ends. Clearly, this DNA itself preferentially forms a hairpin via intrastrand annealing between the lengthened ends, instead of being amplified by the TP. In contrast, the amplification of the primary target DNA is not affected, because it is defined by both the SSP and WP. The feasibility of the bridging PCR has been validated by extending into unknown flanking regions of several known genes. Overall, this bridging PCR could be an alternative to existing genome-walking methods.