Optimization of two-step asymmetric PCR protocol to prepare single-strand DNA for aptamer screening

Aptamers are promising molecular recognition elements. During aptamer screening using the systematic evolution of ligands by exponential enrichment (SELEX) technology, the preparation of single-stranded DNA (ssDNA) is crucial. Asymmetric PCR (A-PCR) is commonly used for ssDNA generation. Due to the complexity of ssDNA library templates and the variability of PCR reagents, optimizing A-PCR is essential to obtain an enriched ssDNA library with high yield and purity.

In this study, the general conditions were initially optimized through single-factor experiments in symmetric PCR. The optimal conditions were 4.8 fmol/µL ssDNA template, 20 µM of each dNTP, 80 nM forward and reverse primers, 2 U Taq DNA polymerase, 1.5 mM Mg²⁺, an annealing temperature of 62 °C, and 14 PCR amplification cycles. Subsequently, two key parameters of A-PCR were optimized through a two-factor cross grouping experiment. The optimized ratio of forward-to-reverse primer and the number of amplification cycles were 150:1 and 36, respectively. The optimized dNTP concentration in A-PCR was 40 µM each.

These results contributed to enhancing the efficiency, purity, and yield of ssDNA preparation. Furthermore, statistical analysis identified key factors as primer concentration and ratio, amplification cycles, Mg²⁺ concentration, and annealing temperature. It is recommended that these critical parameters be prioritized during ssDNA preparation by A-PCR in SELEX procedures.