Lee Ja Yil, Greene Eric C
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.
Methods Mol Biol. 2011;778:243-58. doi: 10.1007/978-1-61779-261-8_16.
Eukaryotic chromosomes are highly packed into chromatin, the basic unit of which is the nucleosome. The presence of nucleosomes and the resulting organization of the genome into higher-order chromatin structures has profound consequences for virtually all aspects of DNA metabolism, including DNA transcription, repair, and chromosome segregation. We have developed novel approaches for nanofabricating "DNA curtains" for high-throughput single-molecule imaging, and we have begun adapting these new research tools in an effort to begin studying chromatin biology at the single-molecule level. In this protocol, we describe procedures for assembly and real-time single-molecule visualization of DNA curtains bound by reconstituted nucleosomes made from recombinant histones.
真核生物染色体高度压缩形成染色质，其基本单位是核小体。核小体的存在以及由此产生的基因组组织成高阶染色质结构，几乎对DNA代谢的所有方面都有深远影响，包括DNA转录、修复和染色体分离。我们开发了用于高通量单分子成像的纳米制造“DNA帘”的新方法，并已开始应用这些新研究工具，以便在单分子水平上研究染色质生物学。在本方案中，我们描述了由重组组蛋白制成的重组核小体结合的DNA帘的组装和实时单分子可视化程序。
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