Quantitative methylation-sensitive arbitrarily primed PCR method to determine differential genomic DNA methylation in...

Relative levels of DNA hypermethylation were quantified in DS individuals using a new method based on a combination of methylation-sensitive arbitrarily primed polymerase chain reaction (MS-AP-PCR) and quantification of DNA fragments with the Agilent 2100 bioanalyzer. Four of the DS individuals had low plasma total homocysteine (tHcy) level (4.3 +/- 0.3 micromol/l) and 4 other had high-tHcy level (14.1 +/- 0.9 micromol/l). Eight healthy control individuals were matched to the DS cases for age, sex, and tHcy levels. We have identified and quantified six hypermethylated fragments. Their sizes ranged from 230-bp to 700-bp. In cases and controls, low-tHcy did not affect methylation level of identified fragments, mean methylation values were 68.0 +/- 39.7% and 52.1 +/- 40.3%, respectively. DNA methylation in DS individuals did not change significantly (59.7+/-34.5%) in response to high-tHcy level in contrast to controls (23.4 +/- 17.7%, P = 0.02). Further, the quantitative MS-AP-PCR using this microfludic system is a useful method for determining differential genomic DNA methylation.