A Cost‐Effective Approach for Single‐Stranded DNA Amplification Using Primer‐Blocked Asymmetric PCR

In vitro amplification of single-stranded oligonucleotide libraries presents a significant challenge due to the potential for excessive byproduct formation. This phenomenon largely affects the quality of the ssDNAs created using the most commonly used methods, e.g., asymmetric PCR, biotin-streptavidin separation, or lambda exonuclease digestion of dsDNA. Here, we describe an improved protocol that combines primer-blocked asymmetric PCR (PBA-PCR) with emulsion PCR and a cost-effective downstream process that altogether alleviates byproduct formation without distorting the sequence space of the ssDNA library. In PBA-PCR, the reaction mixture is complemented with a 3'-phosphate-blocked limiting primer that decreases mispriming, thus reducing polymerization of DNA byproducts. The downstream process includes mixing of the PBA-PCR product with excess reverse complement of the 3'-phosphate-blocked limiting primer and removal of dsDNA strands via biotin-streptavidin separation, yielding purified ssDNAs. In conclusion, we have devised a universally applicable approach for simple and cost-effective production of ssDNA libraries and unique ssDNA sequences with on-demand labeling. Our protocol could be beneficial for a variety of uses, such as generating aptamer libraries for SELEX, creating unique molecular identifiers for a wide range of sequencing applications, providing donor DNA for CRISPR-Cas9 systems, developing scaffold nanostructures, and enabling DNA-based data storage. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Amplification of ssDNA libraries using PBA-PCR Alternate Protocol 1: Amplification of ssDNA libraries using emulsion PBA-PCR with a simplified extraction of PBA-PCR products Basic Protocol 2: Purification of PBA-PCR products to remove dsDNA and conversion of 3'-blocked primer to double-stranded complexes Alternate Protocol 2: Purification of PBA-PCR products to remove both dsDNA and blocking primers from the reaction mixture Support Protocol: Analysis of PBA-PCR products by gel electrophoresis.