Identification of 5-methylcytosine in DNA fragments immobilized on nitrocellulose paper

A method to identify 5-methylcytosine (m5Cyt) in DNA immobilized on nitrocellulose paper by using antibody against m5Cyt raised in rabbits is described. Immobilized restriction fragments of DNA are incubated first with purified antibody against m5Cyt and then with goat anti-rabbit IgG labeled with 125I. Restriction fragments containing m5Cyt are visualized by autoradiography. By using this method, a heavily methylated fragment of about 1700 base pairs was identified in nuclear DNA fom Chinese hamster cells, the methylation pattern of calf thymus satellite I DNA was examined, and chloroplast DNAs that were extracted from various stage of the Chlamydomonas life cycle were compared. Little if any methylation was detected in chloroplast DNA from vegetative cells or from male gametes, whereas homologous DNAs from female gametes and from zygotes were heavily methylated. The sensitivity of the method was examined with calf thymus satellite I DNA (which contains approximately 40 m5Cyt residues per repeat unit of 1400 base pairs) and with phi X174 virion DNA (which contains a single m5Cyt per molecule). The presence of m5Cyt was detected with as little as 40 ng of phi X174 DNA containing 0.02 pmol of m5Cyt and with 100 ng of satellite DNA containing about 0.5 pmol of m5Cyt. Thus, the method makes possible the identification of individual methylated sites in purified DNAs in the size range of single genes.