Highly efficient PCR assay to discriminate allelic DNA methylation status using whole genome amplification
1 School of Electrical and Computer Engineering, College of Science and Engineering, Kanazawa University, Kanazawa 920-1192, Japan
2 Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa 277-8561, Japan
BMC Research Notes 2011, 4:179 doi:10.1186/1756-0500-4-179Published: 10 June 2011
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.
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.
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.