Partially Overlapping Primer-Based PCR for Genome Walking

<div><p>Current genome walking methods are cumbersome to perform and can result in non-specific products. Here, we demonstrate the use of partially overlapping primer-based PCR (POP-PCR), a direct genome walking technique for the isolation of unknown flanking regions. This method exploits the partially overlapping characteristic at the 3’ ends of a set of POP primers (walking primers), which guarantees that the POP primer only anneals to the POP site of the preceding PCR product at relatively low temperatures. POP primer adaptation priming at the genomic DNA/POP site occurs only once due to one low-/reduced-stringency cycle in each nested PCR, resulting in the synthesis of a pool of single-stranded DNA molecules. Of this pool, the target single-stranded DNA is replicated to the double-stranded form bound by the specific primer and the POP primer in the subsequent high-stringency cycle due to the presence of the specific primer-binding site. The non-target single stranded DNA does not become double stranded due to the absence of a binding site for any of the primers. Therefore, the POP-PCR enriches target DNA while suppressing non-target products. We successfully used POP-PCR to retrieve flanking regions bordering the <i>gadA</i> locus in <i>Lactobacillus brevis</i> NCL912, <i>malQ</i> in <i>Pichia pastoris</i> GS115, the human <i>aldolase A</i> gene, and <i>hyg</i> in rice.</p></div

Additional file 1: Table S1. of Genetic engineering of Escherichia coli to improve L-phenylalanine production

Primers used in the genetic engineering. Table S2. The gradient variation ratio of phases A:B in HPLC analysis of L-Phe concentration. Table S3. Primers used in the Real-time PCR analysis. Figure S1. Fermentation consequence of the tyrR knocked out strain. The gray column means OD600 and the black column means L-Phe titer. Figure S2. Sequence alignment results of the TyrRwt and TyrRmut. This result was obtained by DNAMAN software. Figure S3. Overexpression of the precursors’ synthesis encoded genes. The gray column means OD600 and the black column means L-Phe titer. Figure S4. Relative strength of five promoters that were used to overexpress AroD in xllp08. (DOCX 582 kb

Chromosome walking of the <i>gadA</i> locus of <i>Lactobacillus brevis</i> NCL912 (a), human aldolase A gene (b), <i>malQ</i> of <i>Pichia pastoris</i> GS115 (c), and <i>hyg</i> of rice (d).

<p>I: walking into 5’ regions of the genes (locus); II: walking into 3’ regions of the genes (locus). Each walking experiment contained four sets of PCRs that respectively utilized the four POP primer sets, POP1, POP2, POP3, and POP4, paired with a specific primer set. For each set of PCRs, only the results of secondary PCR (left lane) and tertiary (right lane) PCR are presented. White arrows indicate target bands. M1: DL2000 DNA marker. M2: λ-Hind III digest DNA Marker. M3: DL5000 DNA marker.</p