Asynchronous PCR.

Abstract

Asynchronous PCR (aPCR) is a new PCR method that directs an ordered and sequential amplification of the + and - strands of DNA amplicons. There are several unique characteristics of aPCR that generate new application opportunities. The melting temperature (Tm) of the forward and reverse aPCR primers differ by at least 15°C. The concentration of the lower Tm primer is reduced from 900 to 100 nM, thereby allowing for asynchronous or asymmetric strand-specific amplification. Furthermore, unique thermocycling parameter strategy dictates the + and - strand amplification cue. Each aPCR cycle includes two annealing and extension steps. Sequential annealing and extension of forward and reverse primers during each cycle produce transient single-stranded DNA (ssDNA) amplicons which help hybridization-based probes such as peptide nucleic acid (PNA) bind to the target sequences more effectively. This new method can be used in real-time quantitative PCR (qPCR) for gene expression analyses as well as production of robust ssDNA targets for microarray and other hybridization-based applications.