Estimation of the amount of 5-methylcytosine in Drosophila melanogaster DNA by amplified ELISA and photoacoustic spectroscopy.

We have previously reported a sensitive immunochemical method for detecting 5-methylcytosine in DNA which involves spotting DNA samples on nitrocellulose paper and detection of 5-methylcytosine, if any, by a combination of the double antibody method and a staining reaction brought about by biotin-avidin and peroxidase. We report here a linear relationship between the concentration of 5-methylcytosine in DNA and staining intensity, as recorded by photoacoustic spectroscopy. It appears possible to obtain, by this method, reliable quantitative estimates of 5-methylcytosine in nanogram quantities of intact DNA. When Drosophila melanogaster DNA was assayed for the presence of 5-methylcytosine by this method, a faint but clearly positive reaction was obtained. When the photoacoustic intensity of this stained spot is compared with a calibration plot derived from phi X174 DNA whose 5-methylcytosine content is known, we obtain, for D. melanogaster DNA, one 5-methylcytosine residue in approximately 12 500 bases or 0.008 mol% methylation.