DAR-PCR: a new tool for efficient retrieval of unknown flanking genomic DNA

Abstract

Various PCR-based genome-walking methods have been developed to acquire unknown flanking DNA sequences. However, the specificity and efficacy levels, and the operational processes, of the available methods are unsatisfactory. This work proposes a novel walking approach, termed differential annealing-mediated racket PCR (DAR-PCR). The key to DAR-PCR is the use of primer-mediated intra-strand annealing (ISA). An ISA primer consists of a 5’ root homologous to the known sequence and a heterologous 3’ bud. In the single low-stringency cycle, the ISA primer anneals to a site on an unknown region and extends towards the sequence-specific primer (SSP) 1 site, thereby forming a target single-stranded DNA bound by the SSP1 complement and the ISA primer. In the subsequent more stringent cycles, its complementary strand is accumulated, owing to the differential annealing between the moderate-stringency ISA primer and the high-stringency SSP1. The accumulation of this strand provides an opportunity for ISA mediated by the ISA primer root. A loop-back extension subsequent to ISA occurs, creating a racket-like DNA with the known region positioned at both ends of the unknown sequence. This DNA is exponentially amplified during the secondary PCR driven by an SSP pair inner to SSP1. DAR-PCR was validated as an efficient walking method by determining unknown flanking sequences in Lactobacillus brevis and Oryza sativa.

Protocol to retrieve unknown flanking DNA sequences using semi-site-specific PCR-based genome walking

“…Genome-walking is extensively used to probe unknown flanking DNA sequences. 2 , 3 , 4 , 5 , 6 , 7 , 8 Recently, we have devised Wristwatch PCR for genome-walking. 9 The Wristwatch PCR involves a set of three random walking primers that form a wristwatch-like structure with each other.…”

Protocol to access unknown flanking DNA sequences using Wristwatch-PCR for genome-walking

“…Genome-walking is a group of techniques for capturing unknown genomic regions contiguous to a targeted DNA, which is integral to molecular study of chromosomes and significant genetic elements. 2 , 3 As the advancement of molecular biology, genome-walking relying on PCR has been promising given its simplicity and clarity. 4 , 5 According to the involved rationales, the available genome-walking strategies can generally be classified into three types: 1) inverse PCR 6 ; 2) cleavage-ligation-mediated PCR 7 , 8 ; and 3) randomly primed PCR.…”

Sodium-Ion-Free Fermentative Production of GABA with Levilactobacillus brevis CD0817

“…Many target strains have been isolated using these methods [ 15 , 16 , 17 , 18 ]. However, DNA-sequence-based methods are attractive methods in this field [ 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 ].…”