Department of Molecular Biology, Institute of Biochemistry and Molecular Biology, Semmelweis University, Budapest, Hungary.
Curr Protoc. 2024 Sep;4(9):e1125. doi: 10.1002/cpz1.1125.
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.
体外扩增单链寡核苷酸文库是一项重大挑战，因为可能会产生过多的副产物。这种现象在很大程度上影响了使用最常用方法（例如不对称 PCR、生物素-链霉亲和素分离或 dsDNA 的 λ 外切核酸酶消化）创建的 ssDNA 的质量。在这里，我们描述了一种改进的方案，该方案将引物封锁不对称 PCR (PBA-PCR) 与乳液 PCR 以及具有成本效益的下游处理相结合，完全缓解了副产物的形成，而不会扭曲 ssDNA 文库的序列空间。在 PBA-PCR 中，反应混合物中补充了 3'-磷酸封锁的限制引物，该引物可减少错误引发，从而减少 DNA 副产物的聚合。下游处理包括将 PBA-PCR 产物与过量的 3'-磷酸封锁的限制引物的反向互补物混合，并通过生物素-链霉亲和素分离去除 dsDNA 链，从而产生纯化的 ssDNA。总之，我们设计了一种普遍适用的方法，用于简单且具有成本效益的 ssDNA 文库和具有按需标记的独特 ssDNA 序列的生产。我们的方案可用于多种用途，例如为 SELEX 生成适体文库、为广泛的测序应用创建独特的分子标识符、为 CRISPR-Cas9 系统提供供体 DNA、开发支架纳米结构以及实现基于 DNA 的数据存储。© 2024 作者。Wiley Periodicals LLC 出版的《当代协议》。基础方案 1：使用 PBA-PCR 扩增 ssDNA 文库备选方案 1：使用乳液 PBA-PCR 扩增 ssDNA 文库，简化 PBA-PCR 产物的提取基础方案 2：纯化 PBA-PCR 产物以去除 dsDNA 并将 3'-封锁引物转化为双链复合物备选方案 2：从反应混合物中纯化 PBA-PCR 产物以去除 dsDNA 和封锁引物支持方案：通过凝胶电泳分析 PBA-PCR 产物。
