Highly efficient PCR assay to discriminate allelic DNA methylation status using whole genome amplification.
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ABSTRACT: Background
We previously developed a simple method termed HpaII-McrBC PCR (HM-PCR) to discriminate allelic methylation status of the genomic sites of interest, and successfully applied it to a comprehensive analysis of CpG islands (CGIs) on human chromosome 21q. However, HM-PCR requires 200 ng of genomic DNA to examine one target site, thereby precluding its application to such samples that are limited in quantity.Findings
We developed HpaII-McrBC whole-genome-amplification PCR (HM-WGA-PCR) that uses whole-genome-amplified DNA as the template. HM-WGA-PCR uses only 1/100th the genomic template material required for HM-PCR. Indeed, we successfully analyzed 147 CGIs by HM-WGA-PCR using only ~300 ng of DNA, whereas previous HM-PCR study had required ~30 μg. Furthermore, we confirmed that allelic methylation status revealed by HM-WGA-PCR is identical to that by HM-PCR in every case of the 147 CGIs tested, proving high consistency between the two methods.Conclusions
HM-WGA-PCR would serve as a reliable alternative to HM-PCR in the analysis of allelic methylation status when the quantity of DNA available is limited.
SUBMITTER: Yamada Y
PROVIDER: S-EPMC3127961 | biostudies-literature |
REPOSITORIES: biostudies-literature
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