DNA Methylation Analysis of ChIP Products at Single Nucleotide Resolution by Pyrosequencing®

Methods Mol Biol. 2015:1315:315-33. doi: 10.1007/978-1-4939-2715-9_22.

Abstract

Interaction and co-occurrence of protein and DNA-based epigenetic modifications have become a topic of interest for many fundamental and biomedical questions. We describe within this chapter a protocol that combines two techniques in order to determine the methylation status of the DNA specifically associated with a protein of interest. First, DNA that directly interacts with the selected protein (such as a specific histone modification, a transcription factor, or any other DNA-associated protein) is purified by standard chromatin immunoprecipitation (ChIP). Second, the level of DNA methylation of this immunoprecipitated DNA is measured by bisulfite conversion and Pyrosequencing, a quantitative sequencing-by-synthesis method. This procedure allows determining the methylation status of genomic DNA associated to a specific protein at single nucleotide resolution.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Analytic Sample Preparation Methods
  • Cell Line, Tumor
  • Chromatin Immunoprecipitation / methods*
  • DNA / genetics
  • DNA / isolation & purification
  • DNA Methylation* / drug effects
  • DNA Primers / genetics
  • Humans
  • Microspheres
  • Nucleotides / genetics*
  • Polymerase Chain Reaction
  • Promoter Regions, Genetic / genetics
  • Sequence Analysis, DNA / methods*
  • Sulfites / pharmacology

Substances

  • DNA Primers
  • Nucleotides
  • Sulfites
  • DNA
  • hydrogen sulfite