Development of an Optimization Pipeline of Asymmetric PCR Towards the Generation of DNA Aptamer- A Guide for Beginners

Abstract Asymmetric PCR is one of the most utilized strategies in ssDNA generation towards DNA aptamer generation due to its low cost, robustness and the low amount of starting template. Despite its advantages, careful optimization of the asymmetric PCR is still warranted to optimize the yield of ssDNA. In this present study, we have developed an extensive optimization pipeline that involves the optimization of symmetric PCR, optimization of primer amounts/ratios, PCR cycles, annealing temperatures, template concentrations, Mg 2+ /dNTP concentrations and the amounts of Taq Polymerase. To further boost the generation of ssDNA, we have also integrated an additional single-stranded DNA generation method via lambda exonuclease and biotin-streptavidin-based separation into the optimization pipeline to further improve the yield of ssDNA generation. We have acquired 700 ± 11.3 and 820 ± 19.2 nM for A-PCR-lambda exonuclease and A-PCR-biotin-streptavidin-based separation, respectively. We urge to develop a separate optimization pipeline of asymmetric PCR for each different randomized ssDNA library before embarking on any SELEX studies.