Quantitation of genomic methylation using ligation-mediated PCR.
We have developed a new technique for the quantitation of CpG methylation of genomic DNA. This method measures the conversion of a larger amplified DNA fragment to a shorter DNA product correlating with demethylation. The procedure uses pairs of non-isoschizomeric enzymes, one of which is methylation-sensitive, to cleave genomic DNA at closely spaced sites. The extent of cleavage by the methylation-sensitive restriction enzyme is quantitated by amplification of these digestion products with ligation-mediated PCR and radioactive labeling of the product. The ratio of the two amplified fragments correlates with the degree of methylation at the restriction site. The analysis is rapid, quantitative, internally controlled and requires small quantities of genomic DNA.
McGrew
M J
MJ
Boston University School of Medicine.
Rosenthal
N
N
D000818:Animals; D001483:Base Sequence; D003596:Cytosine / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D006147:Guanine / Q000378:metabolism; D008745:Methylation; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009218:Myosins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley
D006147:Guanine; D003596:Cytosine; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C037130:GCGC-specific type II deoxyribonucleases; D009218:Myosins