Rapid and quantitative method of allele-specific DNA methylation analysis
| Autor: | Hui-Lee Wong, Hyang-Min Byun, Jennifer M. Kwan, Mihaela Campan, Sue A. Ingles, Peter W. Laird, Allen S. Yang |
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| Jazyk: | angličtina |
| Rok vydání: | 2006 |
| Předmět: | |
| Zdroj: | BioTechniques, Vol 41, Iss 6, Pp 734-739 (2006) |
| Druh dokumentu: | article |
| ISSN: | 1940-9818 0736-6205 |
| DOI: | 10.2144/000112305 |
| Popis: | 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 Pyrosequencing™. 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 H19 gene, 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. |
| Databáze: | Directory of Open Access Journals |
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