A. BAMCA: MCA method was performed as described by Toyota et al. Samples DNA of non-small cell lung cancer cell line, LK79, was digested with a methylation-sensitive restriction enzyme SmaI, followed by digested with methylation-insensitive XmaI. Adaptors are ligated to XmaI-digested sticky ends, and PCR was performed to amplify the methylation sequences in the presence of Cy3-dCTP. Control DNA is treated in the same manner with Cy5-dCTP. Closed circle indicates Methylated CpGs (marked with Me) and an open circle indicates in unmethylated CpG site.
B, the labeled samples were co-hybridized to MCG Whole Genome Array-4500. Arrays are scanned by use of a GenePix (Axon Instruments, Foster City, CA) fluorescent scanner and analyzed using ArrayGauge software (Axon Instruments). BAMCA with DNA of LK79 shows a positive BAC harboring MINT26 locus (arrow) in the partial image of the array.MINT26 has been reported as a hypermethylated locus in LK79 by Hatada I et al.37)
C, after MINT26 DNA fragments were amplified by PCR, amplicons were digested with SmaI. and electropholesed in 0.4% agarose gel. MINT26 from LK79 is digested with SmaI whereas those from lymphocytes (LC) are not digested, indicating that MINT26 locus is methylated in LK79.