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5C: Detection of Chromosomal Interactions

詳細技術說明

BackgroundThe nucleus of a cell needs to fit alarge amount of DNA into a small area efficiently.  While local chromatin structure has been studied, less isknown about the higher order packing of DNA.  The way chromatin is packaged is a major determinant of acell’s gene expression patterns. Regions of DNA can interact with each otherdespite a large distance from one chromosomal site to another.  These interactions can either enhanceor silence transcription, and classifying these sites on a genomic scale wouldbe an important tool for understanding transcription systems in differentiationor disease states.  Identifyinglong-range interactions between chromatin segments would provide an overview ofthe structural conformation of a genomic region.TechnologyCarbon-copy chromosome conformation capture (5C) isa method of identifying regions of DNA that interact with each other. 3C technologypreviously developed in the Dekker lab allowed for detection of genomicelements interacting at specific genes. 5C builds upon this technique, allowingfor identification of many interactions simultaneously on a genomic scale.  5C combines crosslinking of geneticelements together, synthesizing a library of interacting DNA fragments, andidentifying these fragments by microarray or high-throughput sequencing.Protocol1.    Regionsof chromatin are crosslinked.2.    Thecrosslinked DNA is digested using a restriction enzyme.3.    Interactingregions of DNA are then ligated together.4.    Thecrosslinks are then reversed, creating the 3C library.5.    The3C library is then amplified using tagged universal primers.6.    Thisamplified 5C library is then subjected to microarray or high-throughputsequencing.Applications·     Understandingthe spatial organization of the genome·     Determinationof genome architecture specific to different cell types·     Characterizationof disease states based on interactions of genetic elementsAdvantages·     Highthroughput method of identifying enhancer or repressor elements·     My5Csoftware contains tools designed specifically for 5C design and analysis·     Canbe adapted to kit formatReferencesDekkerlab website: http://3DG.umassmed.eduBaùD, Sanyal A, Lajoie BR, Capriotti E, Byron M, Lawrence JB, Dekker J,Marti-Renom MA.  (2010) Thethree-dimensional folding of the a-globin gene domainreveals formation of chromatin globules. Nat.Struct. Mol. Biol. [Epub ahead of print]Lajoie,B.R., van Berkum, N.L., Sanyal, A. and Dekker, J. (2009) My5C: web tools forchromosome conformation capture studies. Nat. Methods, 6(10): 690-691.vanBerkum, N.L. and Dekker, J. (2009) Determining spatial chromatinorganization of large genomic regions using 5C technology. Methods Mol. Biol., 567: 189-213.Dostie,J., Richmond, T.A., Arnaout, R.A., Selzer, R.R., Lee, W.L., Honan, T.A., Rubio,E.D., Krumm, A., Lamb, J., Nusbaum, C., Green, R.D. and Dekker, J. (2006). ChromosomeConformation Capture Carbon Copy (5C): A massively parallel solution formapping interactions between genomic elements. Genome Research, 16(10): 1299-1309.

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國家/地區

美國