Nanobiotechnology and Bioanalysis group, Department of Chemical Engineering, Universitat Rovira i Virgili, Tarragona, Spain.
Anal Bioanal Chem. 2012 Aug;404(3):835-42. doi: 10.1007/s00216-012-6183-4. Epub 2012 Jun 26.
Single-stranded DNA (ssDNA) generation is a crucial step in several molecular biology applications, such as sequencing or DNA chip and microarray technology. Molecules of ssDNA also play a key role in the selection of ssDNA aptamers through Systematic Evolution of Ligands by EXponential enrichment (SELEX). With particular interest for this application, herein we present a comparative study of the most used methods for generation of ssDNA used in SELEX, such as asymmetric PCR, enzyme digestion and magnetic separation with streptavidin beads. In addition, we evaluate a new technique that combines asymmetric PCR and enzyme digestion with the aim to achieve the maximum efficiency in ssDNA generation. The methods studied were compared in terms of quality of ssDNA using electrophoretic analysis and generated ssDNA yields were quantitatively measured using an Enzyme-Linked OligoNucleotide Assay (ELONA).
单链 DNA（ssDNA）的生成是几种分子生物学应用中的关键步骤，例如测序或 DNA 芯片和微阵列技术。ssDNA 分子在通过指数富集的配体系统进化（SELEX）选择 ssDNA 适体中也起着关键作用。鉴于此应用的特殊兴趣，本文对 SELEX 中用于生成 ssDNA 的最常用方法进行了比较研究，例如不对称 PCR、酶消化和链霉亲和素珠的磁分离。此外，我们评估了一种新技术，该技术将不对称 PCR 和酶消化相结合，旨在实现 ssDNA 生成的最大效率。通过电泳分析比较了所研究的方法在 ssDNA 质量方面的差异，并使用酶联寡核苷酸测定法（ELONA）定量测量了生成的 ssDNA 产量。
