Optimization of DC-PCR for drug screening.

<p>Effect of cytoplasmic enzymes on DC-PCR (<b>A</b>). KMS-12-PE cells were diluted at 1∶25 in hypotonic PCR buffer with or without added protease inhibitors and incubated for 20 min before PCR was run to simulate the time required for pipetting during a small molecule screen. Inclusion of protease inhibitors in the PCR mastermix decreased the number of amplifiable sites in DAC treated and untreated KMS-12-PE cells suggesting that digestion of DNA binding proteins can occur in PCR buffer although epigenetic differences between samples are maintained in the studied example. Detection of DC-PCR product via UV transillumination of PCR plates and sensitivity compared to gel electrophoresis (<b>B</b>). Ethidium bromide was added to the PCR mastermix before serially diluted cells were submitted to DC-PCR: untreated, DAC treated or fibroblasts. After completion of thermal cycling samples were first analyzed under UV light (Left panel), then via agarose-gel electrophoresis (Right panel). Using gel-based detection, the level of detection for epigenetic differences of CDKN2A was lower, down to about 1 cell for site #6 compared to about 300 cells with direct UV transillumination. The primer numbers correspond to sequences on the map described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044690#pone-0044690-g001" target="_blank">Figure 1A</a>. Three independent experiments yielded the same detection levels. Correlation between direct UV transillumination and gel electrophoresis was seen in all experiments (hundreds).