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.
D044503:5-Methylcytosine; D000162:Acoustics; D000818:Animals; D010584:Bacteriophage phi X 174 / Q000235:genetics; D002460:Cell Line; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis; D004247:DNA / Q000032:analysis*; D004279:DNA, Viral / Q000032:analysis; D004331:Drosophila melanogaster / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005333:Fetus; D005347:Fibroblasts / Q000032:analysis; D006801:Humans; D007313:Insecta / Q000235:genetics; D013053:Spectrophotometry
D004279:DNA, Viral; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA