Optimized Asymmetric PCR Protocol Boosts Single-Stranded DNA Production for Aptamer Screening

Researchers have reported an optimized two-step asymmetric PCR protocol for preparing single-stranded DNA, a critical material in the screening of aptamers, which are promising molecular recognition elements. The work, published in Molecular Biology Reports, addresses a practical bottleneck in SELEX, the systematic evolution of ligands by exponential enrichment, where generating an enriched single-stranded DNA library with high yield and purity is essential for successful aptamer discovery.

The team first established general reaction conditions using single-factor experiments in symmetric PCR. Under these optimized conditions, the reaction contained 4.8 fmol/µL single-stranded DNA template, 20 µM of each dNTP, 80 nM of forward and reverse primers, 2 U of Taq DNA polymerase, 1.5 mM Mg2+, an annealing temperature of 62 °C, and 14 amplification cycles.

In the second stage, two key asymmetric PCR parameters were tuned through a two-factor cross grouping experiment. The optimal forward-to-reverse primer ratio was determined to be 150:1, with 36 amplification cycles, and the optimal dNTP concentration in the asymmetric reaction was 40 µM of each nucleotide. Because single-stranded DNA library templates are complex and PCR reagents can vary between preparations, the authors emphasize that such optimization is necessary rather than optional when preparing libraries for SELEX.

Beyond the specific protocol values, statistical analysis of the experiments identified which variables mattered most. Primer concentration and the primer ratio, the number of amplification cycles, Mg2+ concentration, and annealing temperature emerged as key factors. The authors recommend that these critical parameters be prioritized during single-stranded DNA preparation by asymmetric PCR in SELEX procedures.

According to the study, the optimized conditions contribute to enhancing the efficiency, purity, and yield of single-stranded DNA preparation. The findings are presented as an Original Article, with the authors including Huajie Gu, Jiaming Shen, Zihan Chen, Liling Hao, Yuting Liu and Qian Wang.

Subject of Research: Optimization of asymmetric PCR conditions for preparing single-stranded DNA libraries in aptamer screening by SELEX.

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

Article References: Gu, H., Shen, J., Chen, Z., Hao, L., Liu, Y., & Wang, Q. (2026). Optimization of two-step asymmetric PCR protocol to prepare single-strand DNA for aptamer screening. Molecular Biology Reports, 53(1), Article 1849. https://doi.org/10.1007/s11033-026-13068-z

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DOI: 10.1007/s11033-026-13068-z

Keywords: aptamers, SELEX, asymmetric PCR, single-stranded DNA, PCR optimization, molecular biology, primer ratio, DNA polymerase, library preparation, Molecular Biology Reports, Optimization, two-step