Rapid and quantitative method of allele-specific DNA methylation analysis.
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.
Wong
Hui-Lee
HL
University of Southern California, Los Angeles, CA 90089, USA.
Byun
Hyang-Min
HM
Kwan
Jennifer M
JM
Campan
Mihaela
M
Ingles
Sue A
SA
Laird
Peter W
PW
Yang
Allen S
AS
D000483:Alleles; D002460:Cell Line; D019175:DNA Methylation; D005821:Genetic Techniques; D018392:Genomic Imprinting; D006801:Humans; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
C094335:H19 long non-coding RNA; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated