3341 - How are the EpiTect DNA controls produced and tested?
The genomic DNA is derived from disease free human whole blood from multiple anonymous donors. The DNA is pooled after purification to yield a mixture of individuals (male/female) and the whole genome is represented. The whole genome is copied stripping the resultant DNA of methylation.
The methylated DNA control is methylated in vitro using SssI methylase. The methylation efficiency is high but not guaranteed to be 100% for every position. Although methylated under optimized conditions for the enzymatic reaction, some positions are as low as 85% as determined by Pyrosequencing. These sites of lower methylation are not random but rules to predict these low positions are undetermined.QIAGEN checks the consistency of EpiTect Control DNA for methylation and bisulfite conversion using an very sensitive real time MSP-PCR assay. QIAGEN performs several realtime PCR tests to evaluate the bisulfite conversion efficiency as well as the methylation efficiency of the DNAs in the Control DNA Set. We use test systems specific for genomic DNA to detect unconverted DNA or for converted and unmethylated DNA to detect converted but unmethylated DNA, as well as for converted and methylated DNA.