Advanced Medical & Dental Institute (AMDI), Universiti Sains Malaysia, 13200, Bertam, Kepala Batas, Penang, Malaysia.
Faculty of Medicine and Health Sciences, Universiti Malaysia Sarawak (UNIMAS), 93400, Kota Samarahan, Sarawak, Malaysia.
World J Microbiol Biotechnol. 2022 Jan 6;38(2):31. doi: 10.1007/s11274-021-03209-w.
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, Mg/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 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.
不对称 PCR 是用于生成 DNA 适体的 ssDNA 最常用的策略之一，因为它成本低、稳健且起始模板量少。尽管具有这些优势，但仍需要仔细优化不对称 PCR，以优化 ssDNA 的产量。在本研究中，我们开发了一个广泛的优化流程，涉及对对称 PCR 的初始优化，然后对不对称 PCR 进行优化。在不对称 PCR 中，优化了引物量/比例、PCR 循环、退火温度、模板浓度、Mg/dNTP 浓度和 Taq 聚合酶的量。为了进一步提高 ssDNA 的生成效率，我们还将另一种单链 DNA 生成方法，即通过 λ 核酸外切酶或基于生物素-链霉亲和素的分离，整合到优化流程中，以进一步提高 ssDNA 生成的效率。我们分别获得了 700±11.3 和 820±19.2 nM 的 A-PCR-λ 核酸外切酶和 A-PCR-基于生物素-链霉亲和素的分离。我们敦促在进行任何 SELEX 研究之前，为每个不同的随机 ssDNA 文库开发单独的不对称 PCR 优化流程。
