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PMID: 16326863 Published · epublish English Evaluation Study Journal Article Research Support, N.I.H., Extramural

Analysis of repetitive element DNA methylation by MethyLight.

Nucleic acids research ·Vol. 33 ·No. 21 ·2005-00-00 ·Pages 6823-36

Weisenberger DJ, Campan M, Long TI, Kim M, Woods C, Fiala E, Ehrlich M, Laird PW

Abstract

Repetitive elements represent a large portion of the human genome and contain much of the CpG methylation found in normal human postnatal somatic tissues. Loss of DNA methylation in these sequences might account for most of the global hypomethylation that characterizes a large percentage of human cancers that have been studied. There is widespread interest in correlating the genomic 5-methylcytosine content with clinical outcome, dietary history, lifestyle, etc. However, a high-throughput, accurate and easily accessible technique that can be applied even to paraffin-embedded tissue DNA is not yet available. Here, we report the development of quantitative MethyLight assays to determine the levels of methylated and unmethylated repeats, namely, Alu and LINE-1 sequences and the centromeric satellite alpha (Satalpha) and juxtacentromeric satellite 2 (Sat2) DNA sequences. Methylation levels of Alu, Sat2 and LINE-1 repeats were significantly associated with global DNA methylation, as measured by high performance liquid chromatography, and the combined measurements of Alu and Sat2 methylation were highly correlative with global DNA methylation measurements. These MethyLight assays rely only on real-time PCR and provide surrogate markers for global DNA methylation analysis. We also describe a novel design strategy for the development of methylation-independent MethyLight control reactions based on Alu sequences depleted of CpG dinucleotides by evolutionary deamination on one strand. We show that one such Alu-based reaction provides a greatly improved detection of DNA for normalization in MethyLight applications and is less susceptible to normalization errors caused by cancer-associated aneuploidy and copy number changes.

MeSH Terms
Alu Elements Base Sequence Blotting, Southern Consensus Sequence DNA Methylation DNA, Neoplasm/chemistry,metabolism DNA, Satellite Humans Long Interspersed Nucleotide Elements Molecular Sequence Data Polymerase Chain Reaction/methods
Chemicals
DNA, Neoplasm DNA, Satellite
Authors & Affiliations
8 authors, click to expand affiliations / ORCID
Weisenberger Daniel J
Department of Surgery, Keck School of Medicine, USC/Norris Comprehensive Cancer Center, University of Southern California Los Angeles, CA, USA.
Campan Mihaela
Long Tiffany I
Kim Myungjin
Woods Christian
Fiala Emerich
Ehrlich Melanie
Laird Peter W
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
1362-4962
DOI
10.1093/nar/gki987
Published
2005-00-00
Epub
2005-00-02
Pages
6823-36
Language
English
Region
England
NLM ID
0411011
PMCID
PMC1301596
Subset
IM
Grants
NCI NIH HHS · CA 81506 · United States
NCI NIH HHS · R01 CA096958 · United States
NCI NIH HHS · R01 CA075090 · United States
NCI NIH HHS · CA 96958 · United States
NIEHS NIH HHS · R21 ES011672 · United States
NCI NIH HHS · R01 CA081506 · United States
NIEHS NIH HHS · ES 011672 · United States
External Links
PubMed source DOI: 10.1093/nar/gki987 PMC Full Text View journal details
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