Published online 18 February 2004
Nucleic Acids Research, 2004, Vol. 32, No. 3 e38
© 2004 Oxford University Press
A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements
Department of Leukemia, M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA and 1 Department of Biology, IBILCE-UNESP, 2260 Cristovão Colombo, São José do Rio Preto, São Paulo, Brazil
*To whom correspondence should be addressed. Tel: +1 713 745 2260; Fax: +1 713 745 2261; Email: jpissa@mdanderson.org
We report a method for studying global DNA methylation based on using bisulfite treatment of DNA and simultaneous PCR of multiple DNA repetitive elements, such as Alu elements and long interspersed nucleotide elements (LINE). The PCR product, which represents a pool of approximately 15 000 genomic loci, could be used for direct sequencing, selective restriction digestion or pyrosequencing, in order to quantitate DNA methylation. By restriction digestion or pyrosequencing, the assay was reproducible with a standard deviation of only 2% between assays. Using this method we found that almost two-thirds of the CpG methylation sites in Alu elements are mutated, but of the remaining methylation target sites, 87% were methylated. Due to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2'deoxycytidine (DAC), where we found a 1–16% decrease in Alu element and 18–60% LINE methylation within 3 days of treatment. This method can be used as a surrogate marker of genome-wide methylation changes. In addition, it is less labor intensive and requires less DNA than previous methods of assessing global DNA methylation.
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