SIRPH Analysis

SNuPE With IP-RP-HPLC for Quantitative Measurements of DNA Methylation at Specific CpG Sites
  • Osman El-Maarri
Part of the Methods in Molecular Biology™ book series (MIMB, volume 287)

Abstract

This chapter describes a detailed protocol using single-nucleotide primer extension (SNuPE) for quantitative analysis of DNA methylation on specific CpG sites. The first step DNA sample to be studied is treated with sodium bisulfite, which converts selectively unmethylated cytosines to uracil, while methylated cytosines remain unconverted. Subsequently, a SNuPE reaction is performed, with an oligo just flanking a CpG site, using a purified polymerase chain reaction product derived from bisulfite-treated DNA as a template. The oligo is extended by either ddCTP or ddTTP depending on whether the site is methylated or unmethylated, respectively. The reaction is quantitative and linear, and two to three sites can be studied simultaneously in a multiple reaction. The SNuPE product, without further purification, is separated by ion-pair reverse-phase (IP RP) high-performance liquid chromatography (using an alkylated nonporous polysterene-divinylbenzene cartridge) that allows an easy, semiautomated method for separation of the extended and unextended products and an accurate quantification of the extended products. The ratio of the ddCTP to the ddTTP gives the fraction of the methylated cytosines at that specific CpG site.

Key Words

Bisulfite CpG methylation SNuPE DHPLC ion pair reverse-phase HPLC quantitative DNA methylation analysis 

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Copyright information

© Humana Press Inc., Totowa, NJ 2004

Authors and Affiliations

  • Osman El-Maarri
    • 1
  1. 1.Institute of Experimental Haematology and Transfusion MedicineBonnGermany

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