Published online 2 December 2005
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Analysis of repetitive element DNA methylation by MethyLight
Department of Surgery, Department of Biochemistry and Molecular Biology, Keck School of Medicine, USC/Norris Comprehensive Cancer Center, University of Southern California Los Angeles, CA, USA 1Tulane Cancer Center, Human Genetics Program and Department of Biochemistry, Tulane Medical School New Orleans, LA, USA 2Nelson Institute of Environmental Science, New York University School of Medicine Tuxedo, NY, USA
*To whom correspondence should be addressed. Tel: +1 323 865 0650; Fax: +1 323 865 0158; Email: plaird{at}usc.edu
Received August 19, 2005. Revised October 13, 2005. Accepted November 11, 2005.
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 (Sat
) 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.
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