期刊
EPIGENETICS
卷 1, 期 3, 页码 146-152出版社
TAYLOR & FRANCIS INC
DOI: 10.4161/epi.1.3.3392
关键词
DNA methylation; method; restriction enzyme; real-time PCR; gAMP
资金
- Canadian Institutes of Health Research
- Canadian Institutes of Health Research (CIHR)
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.
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