QUANTITATION OF GENOMIC METHYLATION USING LIGATION-MEDIATED PCR

We have developed a new technique for the quantitation of CpG methylat ion of genomic DNA. This method measures the conversion of a larger am plified DNA fragment to a shorter DNA product correlating with demethy lation. The procedure uses pairs of non-isoschizomeric enzymes, one of which is methylation-sensitive, to cleave genomic DNA at closely spac ed sites. The extent of cleavage by the methylation-sensitive restrict ion enzyme is quantitated by amplification of these digestion products with ligation-mediated PCR and radioactive labeling of the product Th e ratio of the two amplified fragments correlates with the degree of m ethylation at the restriction site. The analysis is rapid, quantitativ e, internally controlled and requires small quantities of genomic DNA.