Department of Respiratory Medicine, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori, 036-8562, Japan.
Department of Biochemistry and Genome Biology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori, 036-8562, Japan.
Anal Chim Acta. 2024 May 8;1302:342486. doi: 10.1016/j.aca.2024.342486. Epub 2024 Mar 15.
Analysis of CpG methylation is informative for cancer diagnosis. Previously, we developed a novel method to discriminate CpG methylation status in target DNA by blocking recombinase polymerase amplification (RPA), an isothermal DNA amplification technique, using methyl-CpG binding domain (MBD) protein 2 (MBD2). The method was named MBD protein interference-RPA (MBDi-RPA). In this study, MBDi-RPA was performed using methyl-CpG binding protein 2 (MeCP2), another MBD family protein, as the blocking agent.
MBDi-RPA using MeCP2 detected low levels of CpG methylation, showing that it had higher sensitivity than MBDi-RPA using MBD2. We also developed real-time RPA, which enabled rapid analysis of DNA amplification without the need for laborious agarose gel electrophoresis and used it in combination with MBDi-RPA. We termed this method real-time MBDi-RPA. The method using MeCP2 could determine the abundance ratio of CpG-methylated target DNA simply and rapidly, although highly sensitive detection was challenging.
Real-time MBDi-RPA using MeCP2 could be potentially useful for estimating CpG methylation status in target DNA prior to more detailed analyses.
CpG 甲基化分析对癌症诊断具有重要意义。此前，我们开发了一种新方法，通过使用甲基化-CpG 结合域（MBD）蛋白 2（MBD2）阻断重组酶聚合酶扩增（RPA）这一等温 DNA 扩增技术来区分目标 DNA 中的 CpG 甲基化状态。该方法被命名为 MBD 蛋白干扰-RPA（MBDi-RPA）。在本研究中，我们使用另一种 MBD 家族蛋白甲基化-CpG 结合蛋白 2（MeCP2）作为阻断剂来进行 MBDi-RPA。
使用 MeCP2 的 MBDi-RPA 可以检测到低水平的 CpG 甲基化，表明其比使用 MBD2 的 MBDi-RPA 具有更高的灵敏度。我们还开发了实时 RPA，可以在无需繁琐琼脂糖凝胶电泳的情况下快速分析 DNA 扩增，并将其与 MBDi-RPA 结合使用。我们将这种方法命名为实时 MBDi-RPA。虽然高灵敏度检测具有挑战性，但使用 MeCP2 的方法可以简单快速地确定 CpG 甲基化目标 DNA 的丰度比。
使用 MeCP2 的实时 MBDi-RPA 可能有助于在更详细的分析之前估计目标 DNA 中的 CpG 甲基化状态。
