Singh Badri Nath, Ansari Athar, Hampsey Michael
Department of Biochemistry, Division of Nucleic Acids Enzymology, Robert Wood Johnson Medical School, 683 Hoes Lane West, Piscataway, NJ 08854, USA.
Methods. 2009 Aug;48(4):361-7. doi: 10.1016/j.ymeth.2009.02.018. Epub 2009 Mar 6.
"Chromosome conformation capture" (3C) is a powerful method to detect physical interaction between any two genomic loci. 3C involves formaldehyde crosslinking to stabilize transient interactions, followed by restriction digestion, ligation and locus-specific PCR. Accordingly, 3C reveals complex three-dimensional interactions between distal genetic elements within intact cells at high resolution. Here, we describe a modified 3C protocol designed for detection of transient chromatin interactions in the yeast Saccharomyces cerevisiae. Using this protocol, we are able to detect juxtaposition of promoter and terminator regions of genes with ORFs as short as 1kb in length. We anticipate that this method will be generally applicable to detect dynamic, short-range chromatin interactions and will facilitate the characterization of gene loops and their functional consequences.
“染色体构象捕获”(3C)是一种检测任意两个基因组位点之间物理相互作用的强大方法。3C包括甲醛交联以稳定瞬时相互作用,随后进行限制性消化、连接和位点特异性PCR。因此,3C能在完整细胞内以高分辨率揭示远端遗传元件之间复杂的三维相互作用。在此,我们描述了一种经过改进的3C方案,用于检测酿酒酵母中瞬时染色质相互作用。使用该方案,我们能够检测长度短至1kb的开放阅读框(ORF)基因的启动子和终止子区域的并列情况。我们预计这种方法将普遍适用于检测动态的短程染色质相互作用,并将有助于基因环的表征及其功能后果的研究。