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Nucleic Acids Res. Jun 15, 1997; 25(12): 2532–2534.
PMCID: PMC146738
COBRA: a sensitive and quantitative DNA methylation assay.
Z Xiong and P W Laird
Department of Surgery, University of Southern California School of Medicine, The Norris Comprehensive Cancer Center, Room 6415, Mailstop #73, 1441 Eastlake Avenue, Los Angeles, CA 90033, USA.
We report here on a quantitative technique called COBRA to determine DNA methylation levels at specific gene loci in small amounts of genomic DNA. Restriction enzyme digestion is used to reveal methylation-dependent sequence differences in PCR products of sodium bisulfite-treated DNA as described previously. We show that methylation levels in the original DNA sample are represented by the relative amounts of digested and undigested PCR product in a linearly quantitative fashion across a wide spectrum of DNA methylation levels. In addition, we show that this technique can be reliably applied to DNA obtained from microdissected paraffin-embedded tissue samples. COBRA thus combines the powerful features of ease of use, quantitative accuracy, and compatibility with paraffin sections.
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