Table 1.
Primers used in this study.
Fig 1.
Overview of primer partially overlapping-based PCR.
The first five high-stringency cycles (HSC) of each PCR are to increase copies of the single-stranded DNA of interest. The one low-stringency cycle (LSC) of primary PCR facilitates POP-P annealing to the target DNA and extension towards SP1. The one reduced-stringency cycle (RSC) of secondary PCR allowed POP-S to bind to the POP-P annealing site. A double-stranded target molecule was synthesized in the first HSC following LSC/RSC, and served as the template for the remaining HSCs; non-specific amplification was inhibited because the double-stranded form could not be obtained from a non-target single strand. Solid lines: the known sequence; dotted lines: the unknown sequence; thick black arrows with different heads: nested, specific primers; hollow arrows with different tails: POP primers; gray arrows: primers complementary sequences.
Fig 2.
Chromosome walking of the gadA locus of Lactobacillus brevis NCL912 (a), human aldolase A gene (b), malQ of Pichia pastoris GS115 (c), and hyg of rice (d).
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.