Figure 3

Methylation assessed by sequencing. Representative results of cloning and sequencing of ten genes. COBRA PCR products were cloned into PGem T-easy vector and colonies were picked and inserts sequenced. Each line is representative of an individual allele. Each circle represents a single CpG dinucleotide, filled circles represent a methylated CpG dinucleotide whereas clear circles represent an unmethylated CpG dinucleotide. The MI was calculated as a percentage using the equation; number of CpG dinucleotides methylated/total number of CpG dinucleotides sequenced x 100. Sequencing of the DNA from control bone marrow shows no or very infrequent methylation, with MIs all below 3.5%. For each gene two samples for which COBRA analysis detected hypermethylation and two for which it did not were sequenced. The figure shows control bone marrow and two methylated samples for each gene. The sequencing results correlate with the COBRA detected methylation.