Department of Chemistry, The Pennsylvania State University, University Park, PA 16802, USA.
Anal Biochem. 2013 Apr 15;435(2):181-6. doi: 10.1016/j.ab.2013.01.008. Epub 2013 Jan 31.
Single-stranded DNA (ssDNA) ligation is a crucial step in many biochemical assays. Efficient ways of carrying out this reaction, however, are lacking. We show here that existing ssDNA ligation methods suffer from slow kinetics, poor yield, and severe nucleotide preference. To resolve these issues, we introduce a hybridization-based strategy that provides efficient and low-bias ligation of ssDNA. Our method uses a hairpin DNA to hybridize to any incoming acceptor ssDNA with low bias, with ligation of these strands mediated by T4 DNA ligase. This technique potentially can be applied in protocols that require ligation of ssDNA, including ligation-mediated polymerase chain reaction (LMPCR) and complementary DNA (cDNA) library construction.
单链 DNA（ssDNA）连接是许多生化分析中的关键步骤。然而，缺乏有效的方法来进行这种反应。我们在这里表明，现有的 ssDNA 连接方法存在动力学缓慢、产量低和严重的核苷酸偏好等问题。为了解决这些问题，我们引入了一种基于杂交的策略，该策略提供了 ssDNA 的高效和低偏差连接。我们的方法使用发夹 DNA 以低偏差与任何进入的受体 ssDNA 杂交，这些链的连接由 T4 DNA 连接酶介导。该技术可能适用于需要 ssDNA 连接的方案，包括连接介导的聚合酶链反应（LMPCR）和 cDNA 文库构建。
