Assessment of Changes in Global DNA Methylation Levels by Pyrosequencing® of Repetitive Elements

Methods Mol Biol. 2015;1315:201-7. doi: 10.1007/978-1-4939-2715-9_15.

Abstract

Transposable elements (TE) comprise half of the human genome. LINE-1 and ALU are the most common TE, and they have been used to assess changes in the DNA methylation of repetitive elements in response to intrinsic and extrinsic cellular events. Pyrosequencing(®) is a real-time sequencing technology that enables quantitative assessment of TE methylation at single-base resolution. In Pyrosequencing, a region of interest is first amplified from bisulfite-converted DNA by polymerase chain reaction (PCR), before PCR amplicons are rendered single stranded and annealed with the Pyrosequencing primer prior to sequencing. In this chapter, we provide an overview of the analysis of repetitive element DNA methylation by bisulfite Pyrosequencing, and we describe a protocol that can be used for such purposes.

MeSH terms

  • Analytic Sample Preparation Methods
  • DNA Methylation / drug effects
  • DNA Methylation / genetics*
  • DNA Primers / genetics
  • DNA Transposable Elements / genetics*
  • Genome, Human / genetics
  • Genomics / methods*
  • Humans
  • Sequence Analysis, DNA / methods*
  • Sulfites / pharmacology

Substances

  • DNA Primers
  • DNA Transposable Elements
  • Sulfites
  • hydrogen sulfite