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Rapid and quantitative method of allele-specific DNA methylation analysis
- Source :
- Biotechniques; December 2006, Vol. 41 Issue: 6 p734-739, 6p
- Publication Year :
- 2006
-
Abstract
- Several biological phenomena depend on differential methylation of chromosomal strands. While understanding the role of these processes requires information on allele-specific methylation, the available methodologies are not quantitative or labor-intensive. We describe a novel, rapid method to quantitate allele-specific DNA methylation based on the combination of bisulfite PCR and Pyrosequencingâ„¢. In this method, DNA is first treated with sodium bisulfite, which converts cytosine but not 5-methylcytosine to uracil. Genes of interest are subsequently amplified using PCR. Allele-specific methylation can then be determined by pyrosequencing each allele individually using sequencing primers that incorporate single nucleotide polymorphisms (SNPs) that allow differentiation between the two parental alleles. This allele-specific methylation methodology can potentially afford quantitative analyses relevant to the regulation of X chromosome inactivation, allele-specific expression of genes in the immune system, repetitive elements, and genomic imprinting. As an illustration of our new method, we quantitated allele-specific methylation of the differentially methylated region of the H19gene, which is imprinted. Although we could reliably determine allele-specific methylation with our technique, additional studies will be required to confirm the ability of our assay to measure loss of imprinting.
Details
- Language :
- English
- ISSN :
- 07366205
- Volume :
- 41
- Issue :
- 6
- Database :
- Supplemental Index
- Journal :
- Biotechniques
- Publication Type :
- Periodical
- Accession number :
- ejs45636883
- Full Text :
- https://doi.org/10.2144/000112305