A Novel Method, PCR-Mediated Amplification of Methylated Sites, Detects Methylated CpG Islands in the Genome of Bombyx mori

Abstract

An amplification-mediated method for the analysis of the methylation status was designed and applied to Bombyx mori DNA. The current method, named PAM, utilized the differential methylation sensitivities of four-base cutters, HapII and MspI. In order to reduce background signals, the first HapII digestion step was followed by the ligation to the HapII adaptor, while the second digestion by the same enzyme was followed by the Klenow-filling of the 5′ protruding ends. Then comes the step of digestion with either MspI or HapII, and further both digests were subjected to ligation with the MspI adaptor and to PCR with primers annealing to the MspI adaptor. The PCR products found in the MspI digest, not in the HapII digest, are expected to represent specifically the DNA sequences flanked by the two methylated CpGs in the context of CCGG. In fact, these types of specific signals were given by more than twenty fragments derived from DNA of a moth. By adding another primer that anneals to the HapII adaptor at the PCR step allowed the detection of DNA fragments neighbored by a methylated site at either of the sides. The PAM method using genomic DNA of five larval tissues exhibited differences in the methylation pattern among them. These findings indicate that the PAM method is a powerful tool for screening genome-wide methylation status in the B. mori cells.