Fig. 1

Outline of stepwise partially overlapping primer-based PCR. The first five high-stringency cycles (HSCs) of each PCR were carried out to increase the copy number of specific single-stranded DNA (ssDNA) of interest with different length. The one low-stringency cycle (LSC) of primary PCR allowed SWPOP-P to anneal to the target DNA and extend towards SP-P. The one reduced-stringency cycle (RSC) of secondary/tertiary PCR allowed SWPOP-S/SWPOP-T to bind to the prior SWPOP complementary site(s). A double-stranded target DNA molecule was obtained in the first HSC following LSC/RSC, and served as the template for the remaining twenty-four HSCs. Non-target amplification was suppressed because the double-stranded form could not be synthesized from a non-target single strand. Drawings on the right side: potential nonspecific (non-target) amplifications; SP-P, SP-S, and SP-T: specific primer for primary, secondary, and tertiary PCR, respectively; SWPOP-P, SWPOP-S, and SWPOP-T: stepwise partially overlapping primer for primary, secondary, and tertiary PCR, respectively; solid lines: the known sequence; dotted lines: the unknown sequence; grey arrows: primers complementary sites