Figure 5

DNA replication is not affected by DNA methylation. As shown in Panels A, B, and C, the results of three independent tests using different portions of yeast genomic DNA (which is naturally unmethylated [38]) provide evidence that methylation does not affect PCR results. In these experiments, random PCR was performed using one of the primers (pfm 3 (5'-cy3-dCTGGATAGCGTC), pfm 10 (5'-cy3-dGCGCATTAGACG) and pfm 12 (5'-cy3-dAGAACGCGCCTG)) with Taq DNA polymerase. (Random PCR is a variation of PCR employing only a single primer and performed at a lower annealing temperature [~26°C], generating primer sequence-independent DNA fragments [31]). Lane 1 is a100-bp size marker. Bands indicated by α and β (in lane 2 of panels A, B and C) are DNA fragments containing HpaII methylation/restriction site(s), as their cleavage resulted in their disappearance from lane 4. The presence of α and β bands in lane 2 in panels A, B and C demonstrate that these regions could be amplified by random PCR even though they contained a methylation site.