A sensitive new method for rapid detection of abnormal methylation patterns in global DNA and within CpG islands.
To assess alterations in DNA methylation density in both global DNA and within CpG islands, we have developed a simple method based on the use of methylation-sensitive restriction endonucleases that leave a 5' guanine overhang after DNA cleavage, with subsequent single nucleotide extension with radiolabeled [(3)H]dCTP. The methylation-sensitive restriction enzymes HpaII and AciI have relatively frequent recognition sequences at CpG sites that occur randomly throughout the genome. BssHII is a methylation sensitive enzyme that similarly leaves a guanine overhang, but the recognition sequence is nonrandom and occurs predominantly at unmethylated CpG sites within CpG islands. The selective use of these enzymes can be used to screen for alterations in genome-wide methylation and CpG island methylation status, respectively. The extent of [(3)H]dCTP incorporation opposite the exposed guanine after restriction enzyme treatment is directly proportional to the number of unmethylated (cleaved) CpG sites. The "cytosine-extension assay" has several advantages over existing methods because (a) radiolabel incorporation is independent of the integrity of the DNA, (b) methylation detection does not require PCR amplification or DNA methylase reactions, and (c) it is applicable to ng quantities of DNA. Using DNA extracted from normal human liver and from human hepatocellular carcinoma, the applicability of the assay is demonstrated by the detection of an increase in genome-wide hypomethylation and CpG island hypermethylation in the tumor DNA.
Pogribny
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Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas, 72079, USA.
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James
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D006528:Carcinoma, Hepatocellular; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D019175:DNA Methylation; D004262:DNA Restriction Enzymes; D004273:DNA, Neoplasm / Q000737:chemistry* / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000032:analysis*; D006801:Humans; D008113:Liver Neoplasms; D015183:Restriction Mapping / Q000379:methods*; D012680:Sensitivity and Specificity; D014316:Tritium; D014407:Tumor Cells, Cultured
D004247:DNA; D004262:DNA Restriction Enzymes; D004273:DNA, Neoplasm; D003845:Deoxycytosine Nucleotides; D015226:Dinucleoside Phosphates; D014316:Tritium; C024107:2'-deoxycytidine 5'-triphosphate; C015772:CpG dinucleotide