Chromosome Conformation Capture (3C) in Budding Yeast

Cold Spring Harb Protoc. 2015 Jun 1;2015(6):580-6. doi: 10.1101/pdb.prot085175.

Abstract

Chromosome conformation capture (3C) is a method for studying chromosomal organization that takes advantage of formaldehyde cross-linking to measure the spatial association of two pieces of chromatin. The 3C method begins with whole-cell formaldehyde fixation of chromatin. After cell lysis, solubilized chromatin is digested with a type II restriction endonuclease, and cross-linked DNA fragments are ligated together. Cross-links are reversed by degradation with proteinase K, and chimeric DNA molecules are purified by standard phenol:chloroform extraction. The resulting 3C library represents chromatin fragments that may be separated by large genomic distances or located on different chromosomes, but are close enough in three-dimensional space for cross-linking. Locus-specific oligonucleotide primers are used to detect interactions of interest in the 3C library using end-point polymerase chain reaction (PCR).

MeSH terms

  • Chromatin / chemistry
  • Chromatin / metabolism*
  • Chromosomes / drug effects
  • Cross-Linking Reagents / pharmacology
  • Formaldehyde / pharmacology
  • Molecular Conformation*
  • Saccharomycetales / ultrastructure*

Substances

  • Chromatin
  • Cross-Linking Reagents
  • Formaldehyde