Identification and quantification of differentially methylated loci by the pyrosequencing technology.

Emelyne Dejeux*, Hafida El abdalaoui, Ivo Glynne Gut, Jörg Tost

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

51 Scopus citations

Abstract

Most available protocols for gene-specific DNA methylation analysis are either labor intensive, not quantitative, or limited to the measurement of the methylation status of only one or very few CpG positions. Pyrosequencing is a real-time sequencing technology that overcomes these limitations. After bisulfite modification of genomic DNA, a region of interest is amplified by polymerase chain reaction (PCR) with one of the two primers being biotinylated. The PCR-generated template is rendered single stranded and a pyrosequencing primer is annealed to analyze quantitatively CpGs within 120 bases. Advantages of the pyrosequencing technology are the ease of its implementation, the high quality and the quantitative nature of the results, and its ability to identify differentially methylated positions in close proximity. A minimum amount of 10 ng of bisulfite-treated DNA is necessary to obtain high reproducibility and avoid random amplification. The required DNA amount can be provided by an individual sample or a pool of samples to rapidly investigate the presence of variable DNA methylation patterns. The use of pools and serial pyrosequencing, that is, the successive use of several pyrosequencing primers on the same DNA template, significantly reduces cost, labor, and analysis time as well as saving precious DNA samples for the analysis of gene-specific DNA methylation patterns.

Original languageEnglish (US)
Pages (from-to)189-205
Number of pages17
JournalMethods in molecular biology (Clifton, N.J.)
Volume507
StatePublished - Jan 1 2009

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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