Enzymatic regional methylation assay: a novel method to quantify regional CpG methylation density.

Abstract:

:We have developed a novel quantitative method for rapidly assessing the CpG methylation density of a DNA region in mammalian cells. After bisulfite modification of genomic DNA, the region of interest is PCR amplified with primers containing two dam sites (GATC). The purified PCR products are then incubated with 14C-labeled S-adenosyl-L-methionine (SAM) and dam methyltransferase as an internal control to standardize DNA quantity. This is followed by an incubation with 3H-labeled SAM and SssI methyltransferase for methylation quantification. By use of standard mixtures of cell line DNA with a defined methylation status in every assay, the ratio (3H/14C signal) of each sample can be converted into percentage values to assess the methylation density of the amplified sequence. This methylation-sensitive technique, termed ERMA (Enzymatic Regional Methylation Assay) provides several advantages over existing methods used for methylation analysis as it determines an exact measurement of the methylation density of the region studied. We demonstrate a use of this technique in determining the methylation density of the promoter region of the tumor suppressor gene p15INK4B and changes that occur after treatment with demethylating agents.

journal_name

Genome Res

journal_title

Genome research

authors

Galm O,Rountree MR,Bachman KE,Jair KW,Baylin SB,Herman JG

doi

10.1101/gr.202501

subject

Has Abstract

pub_date

2002-01-01 00:00:00

pages

153-7

issue

1

eissn

1088-9051

issn

1549-5469

journal_volume

12

pub_type

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