Evaluation of a quantitative DNA methylation analysis technique using methylation-sensitive/dependent restriction enzymes and real-time PCR.
DNA methylation in mammals has been shown to play many important roles in diverse biological phenomena. Several methods have been developed for the measurement of region-specific levels of DNA methylation. We sought a technique that could be used to quantitatively evaluate multiple independent loci in several tissues in a quick and cost-effective manner. Recently, a few quantitative techniques have been developed by employing the use of real-time PCR, though they require the additional step of sodium bisulfite conversion. Here we evaluate a technique that involves the digestion of non-sodium bisulfite-treated genomic DNA using methylation-sensitive and methylation-dependent restriction enzymes followed by real-time PCR. The utility of this method is tested by analyzing seventeen genomic regions of known tissue-specific levels of DNA methylation including three imprinted genes. We find that this approach generates rapid, reproducible and accurate results (range = +/-5%) without the additional time required for bisulfite conversion. This approach is also adaptable for use with smaller amounts of starting material. We propose this method as a rapid, quantitative method for the analysis of DNA methylation at single sites or within small regions of DNA.
Oakes
Christopher C
CC
Department of Pharmacology and Therapeutics, The Montreal Children's Hospital Research Institute, McGill University, Montreal, Quebec, Canada.
La Salle
Sophie
S
Robaire
Bernard
B
Trasler
Jacquetta M
JM
D000818:Animals; D001921:Brain / Q000502:physiology; D019175:DNA Methylation; D017931:DNA Primers; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000378:metabolism*; D015226:Dinucleoside Phosphates; D018392:Genomic Imprinting; D007422:Intestines / Q000502:physiology; D008099:Liver / Q000502:physiology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D013698:Templates, Genetic; D013737:Testis / Q000502:physiology
D017931:DNA Primers; D004262:DNA Restriction Enzymes; D015226:Dinucleoside Phosphates; C015772:CpG dinucleotide