爪蟾卵提取物中单分子 DNA 复制的分析。
Single-molecule analysis of DNA replication in Xenopus egg extracts.
机构信息
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
出版信息
Methods. 2012 Jun;57(2):179-86. doi: 10.1016/j.ymeth.2012.03.033. Epub 2012 Apr 6.
Abstract
The recent advent in single-molecule imaging and manipulation methods has made a significant impact on the understanding of molecular mechanisms underlying many essential cellular processes. Single-molecule techniques such as electron microscopy and DNA fiber assays have been employed to study the duplication of genome in eukaryotes. Here, we describe a single-molecule assay that allows replication of DNA attached to the functionalized surface of a microfluidic flow cell in a soluble Xenopus leavis egg extract replication system and subsequent visualization of replication products via fluorescence microscopy. We also explain a method for detection of replication proteins, through fluorescently labeled antibodies, on partially replicated DNA immobilized at both ends to the surface.
摘要
近年来，单分子成像和操控方法的出现对理解许多基本细胞过程的分子机制产生了重大影响。单分子技术，如电子显微镜和 DNA 纤维分析，已被用于研究真核生物基因组的复制。在这里，我们描述了一种单分子检测方法，该方法允许在可溶性非洲爪蟾卵提取物复制系统中附着在微流控流动池功能化表面上的 DNA 进行复制，并通过荧光显微镜观察复制产物。我们还解释了一种方法，用于通过荧光标记的抗体检测部分复制的 DNA 两端固定在表面上的复制蛋白。
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