Development of an optimization pipeline of asymmetric PCR towards the generation of DNA aptamers: a guide for beginners

Tzi, Shien Yeoh and Anna, Andrew and Tang, Thean‑Hock and Marimuthu, Citartan (2022) Development of an optimization pipeline of asymmetric PCR towards the generation of DNA aptamers: a guide for beginners. World Journal of Microbiology and Biotechnology, 38 (31). pp. 1-13. ISSN 1573-0972

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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 initially followed by the optimization of asymmetric PCR. In the asymmetric PCR, optimization of primer amounts/ratios, PCR cycles, annealing temperatures, template concentrations, Mg2+/dNTP concentrations and the amounts of Taq Polymerase was carried out. To further boost the generation of ssDNA, we have also integrated an additional single-stranded DNA generation method, either via lambda exonuclease or biotinstreptavidin-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 diferent randomized ssDNA library before embarking on any SELEX studies.

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