Figure 1

Sensitive-melting analysis after real time-methylation specific PCR (SMART-MSP) assessment of DAPK1 . Gel electrophoresis of representative SMART-MSP products in duplicate, assessing methylation of the DAPK1 promoter 1 region. High resolution melting curve analysis (MCA) is represented by + (indicating methylation) and - (indicating no methylation). Quantification of methylation is reported in %. (100% - fully methylated control, 0.1% - 0.1% dilution control, WGA - unmethylated control, NTC – non-template control). The band sizes observed for the 100% methylated control and 0.1% control were appropriately of identical size, due to the inability of this methodology to differentiate quantities of methylation. Sample 6-7 and sample 6-6 demonstrated the presence of methylation on MCA, with gel electrophoresis of PCR products producing band sizes similar to the methylated controls. However, when quantified, the levels of methylation measured below 0.1%. For sample 6-9 and sample 5-5, bands similar to the methylated controls were observed, despite MCA and quantification indicating no detectable levels of methylation. These bands represent false positive results.