Date Published: May 9, 2018
Publisher: Springer Berlin Heidelberg
Author(s): Kunpeng Chang, Qiong Wang, Xiaofei Shi, Shuixing Wang, Hongjing Wu, Lijuan Nie, Haixing Li.
A stepwise partially overlapping primer-based PCR (SWPOP-PCR) method for isolating flanking unknown DNA regions was developed, which comprises three rounds of nested PCRs sequentially driven by SWPOP primer-nested specific primer pairs. SWPOP primer set is characterized by a partial overlap of 10 bp with 3′-part of the latter primer is identical to 5′-part of the former one, which makes the SWPOP primer in use anneal to SWPOP site of the prior PCR product only at relatively low temperature. For each PCR, target single-stranded DNA primed by the SWPOP primer in the exclusive one low-stringency cycle is converted into double-stranded form in the following high-stringency cycle due to the presence of a perfect annealing site for the specific primer. This double-stranded DNA bounded by the specific primer and the SWPOP primer is exponentially amplified in the remaining high-stringency cycles. Non-target single-stranded DNA, however, cannot be amplified given the lack of perfect complementary sequences for any primers. Therefore, the partial overlap of a SWPOP primer set preferentially synthesizes target products but inhibits nonspecific amplification. We successfully exploited SWPOP-PCR to obtain the DNA sequences flanking glutamate decarboxylase gene (gadA) locus in Lactobacillus brevis NCL912 and hygromycin gene (hyg) integrated in rice.
Numerous PCR-based genome walking methodologies have been developed for identification and isolation of neighboring unknown DNA sequences adjacent to known genomic regions, which can be classified into three main categories (Kotik 2009; Leoni et al. 2011): (I) inverse PCR (Ochman et al. 1988); (II) ligation mediated PCR (Mueller and Wold 1989; Arnold and Hodgson 1991; Jones and Winistorfer 1992; Yan et al. 2003; Ji and Braam 2010); and (III) randomly primed PCR (Liu and Whittier 1995; Tan et al. 2005; Wang et al. 2013). The first two categories rely on labor-intensive and time-consuming restriction digestion and ligation of genomic DNA before PCR amplification (Rosenthal and Jones 1990; Acevedo et al. 2008; Leoni et al. 2008, 2010; Trinh et al. 2012b; Spalinskas et al. 2013). In addition, requirements of high-quality genomic DNA and several different restriction enzymes limit the actual utilization of these methods (Iwahana et al. 1994; Tsuchiya et al. 2009; Bae and Sohn 2010; Trinh et al. 2012a). The third category requires no complicated DNA manipulations before or after PCR (Liu and Chen 2007; Luo et al. 2011). However, the excessive accumulation of non-target DNA products as a consequence of nonspecific annealing of arbitrary primer is the major limitation of these methods (Terauchi and Kahl 2000; Reddy et al. 2008; Thirulogachandar et al. 2011).
The key to the success of the proposed method is to have a possible annealing site in the unknown DNA sequence for SWPOP-P primer (SWPOP primer for primary PCR). In our PCR method, a SWPOP-P primer should find an adapted site in the plate given the fact that one super low-stringency (25 °C) cycle was performed in primary PCR. To increase the success rate in genomic walking, we designed four parallel SWPOP-P primers having heterologous 15 bp at the 3′-parts and identical 10 bp at the 5′-parts. The four heterologous 3′-parts are expected to guarantee at least one SWPOP-P primer creates an annealing site adapted for this SWPOP-P within genomic DNA (Parker et al. 1991; Parks et al. 1991).