A simple modification increases specificity and efficiency of asymmetric PCR

Area of Science:

• Molecular Biology
• Biotechnology

Background:

• In vitro production of single-stranded DNA (ssDNA) is crucial for molecular biology but remains challenging.
• Existing methods struggle to meet the demand for high-purity ssDNA.

Purpose of the Study:

• To develop a novel method for efficient and cost-effective in vitro production of high-purity ssDNA.
• To address challenges in ssDNA synthesis, including mispriming and PCR bias.

Main Methods:

• Primer-blocked asymmetric PCR (PBA-PCR) was developed, utilizing a 3' phosphate-blocked limiting primer.
• High-throughput sequencing was employed to analyze the efficiency and bias of PBA-PCR.
• The method's applicability was validated using SELEX (Systematic Evolution of Ligands by Exponential Enrichment) and aptamer screening assays.

Main Results:

• PBA-PCR selectively produces the desired ssDNA, minimizing DNA by-products caused by mispriming.
• The method effectively amplifies DNA libraries across a large sequence space without introducing significant bias.
• PBA-PCR demonstrated practical utility in SELEX and aptamer candidate screening.

Conclusions:

• PBA-PCR is a generally applicable, straightforward, and cost-effective method for producing high-purity ssDNA.
• The technique alleviates PCR bias, ensuring accurate representation of sequence space.
• PBA-PCR facilitates on-demand labeling of ssDNA for various molecular biology applications.