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Research Paper
Evaluation of a Quantitative DNA Methylation Analysis Technique using Methylation-Sensitive/Dependent Restriction Enzymes and Real-Time PCR
Christopher C Oakes, Sophie La Salle, Bernard Robaire and Jacquetta M Trasler
volume 1 | issue 3
july/august/september 2006Pages: 146 - 152
This is an open-access article
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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.
Authors
Christopher C Oakes
McGill University, Montreal, Quebec, Canada
Sophie La Salle
McGill University, Montreal, Quebec, Canada
Bernard Robaire
McGill University, Montreal, Quebec, Canada
Jacquetta M Trasler
McGill University, Montreal, Quebec, Canada
This is an open-access article
Download PDFIf the document does not open, please right-click on the link (control-click on a Macintosh) and select the option to save the file to disk.