COBRA: a sensitive and quantitative DNA methylation assay.
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.
Xiong
Z
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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.
Laird
P W
PW
D001483:Base Sequence; D015179:Colorectal Neoplasms / Q000737:chemistry / Q000473:pathology; D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000737:chemistry*; D015226:Dinucleoside Phosphates / Q000032:analysis / Q000737:chemistry; D015894:Genome, Human; D006801:Humans; D007202:Indicators and Reagents; D016133:Polymerase Chain Reaction / Q000379:methods; D011960:Receptors, Estrogen / Q000235:genetics*; D015183:Restriction Mapping / Q000379:methods*; D012680:Sensitivity and Specificity; D013447:Sulfites
D004247:DNA; D004273:DNA, Neoplasm; D015226:Dinucleoside Phosphates; D007202:Indicators and Reagents; D011960:Receptors, Estrogen; D013447:Sulfites; C015772:CpG dinucleotide; C009279:sodium bisulfite