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

Abstract 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