染色体互作研究3C35.ppt

Carbon-Copy Chromosome Conformation Capture (5C) (5C) combines 3C with ligation‐mediated amplification (LMA) to simultaneously quantify hundreds of thousands of physical DNA contacts by microarray or ultra‐high‐throughput DNA sequencing. 5C allows the mapping of extensive networks of physical interactions among large sets of genomic elements throughout the genome. 基因互作的研究方法 3C,4C,5C 3C(Chromosome conformation capture) 染色体构象俘获技术 通过一种定量手段（PCR产物的有\无，量的高\低）对DNA之间是否存在相互作用进行研究 过程：甲醛交联，限制性酶切，稀释和连接，解交联，DNA纯化，PCR鉴定） 3C 3C methodology was developed to study spatial organization of long genomic regions in living cells. chromatin is fixed with formaldehyde in vivo to cross-link interacting sites, digested with a restriction enzyme and ligated at a low DNA concentration . Ligation products are then analyzed and quantified by PCR. There are two major types of ligation junctions that are over-represented. One is the junction that forms between neighboring DNA fragments due to incomplete digestion, which represents about 20-30% of all junctions. The other is the junction that forms when one end of the fragment ligates with the other end of the same fragment, and contributes up to 30% of all junctions formed. Circularized Chromosome Conformation Capture (4C) a significant advantage over 3C in that only the sequence of one of the site of interest needs to be known. The fragment, known as the “bait”, contains the site that associates with other chromosomal regions.