Department of Applied Chemistry, Faculty of Science and Engineering, Toyo University, 2100 Kujirai, Kawagoe, Saitama, 350-8585, Japan.
Department of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, 2-8-1 Mejirodai, Bunkyo, Tokyo, 112-8681, Japan.
Anal Biochem. 2020 Mar 15;593:113596. doi: 10.1016/j.ab.2020.113596. Epub 2020 Jan 24.
We present a mechanistic investigation of bead-based padlock rolling circle amplification (RCA) under molecular crowding conditions, a sensitive and selective DNA detection method we have developed. Several important points to optimize the method were clarified: (i) the increase in the number of RCA products is proportional to the excluded volume of poly(ethylene glycol) (PEG), (ii) PEG facilitates ligation of padlock probe to form circular concatemers and monomers, both of which may act as a template for RCA, and (iii) hybridization of detection probe to the products may be facilitated at higher PEG concentrations.
我们提出了基于珠的发夹环滚环扩增（RCA）在分子拥挤条件下的机制研究，这是我们开发的一种灵敏和选择性的 DNA 检测方法。我们已经阐明了几个优化该方法的要点：（i）RCA 产物的数量与聚乙二醇（PEG）的排除体积成正比，（ii）PEG 促进发夹探针的连接形成环状连接物和单体，两者都可以作为 RCA 的模板，以及（iii）在较高的 PEG 浓度下，检测探针与产物的杂交可能更容易。
