Figure 3.

Quantitative ability of MS-qFRET. (A) Experiments with different ratios of unmethylated and methylated DNA (see Methods) show that increasing percent CDKN2A methylation levels are accompanied by an increase in acceptor (Cy5) emission at 670 nm and corresponding donor (QD605) quenching at 605 nm. (B) q-scores are plotted for the varying levels of CDKN2A methylation and a linear fit is observed with r² = 0.999. Results are plotted from five separate mixing experiments. (C) Quantitative ability of MS-qFRET to estimate CDKN2A methylation reversal in DNA from RKO cells treated with DAC for different time points. Error bars are generated from three separate repetitions. q-scores indicate a drop in the level of methylation post-treatment. (D) Quantification of methylation reversal at CDKN2B using MS-qFRET in six MDS patients using 150 ng of input DNA during their first cycle of epigenetic therapy. Changes in levels of methylation are effectively captured to show varying cellular responses to therapy with 5-azacytidine and MS-275.