Improved recovery of bisulphite-treated cell-free DNA in plasma

Detection of cell-free methylated DNA in plasma is a promising tool for tumour diagnosis and monitoring. Due to the very low amount of cell-free DNA in plasma, sensitivity of the detection methods are of utmost importance. The vast majority of currently available methods for analysing DNA methylation are based on bisulphite-mediated deamination of cytosine. However, the recovery of bisulphite-converted DNA is very poor. Here we introduce an alternative method for the crucial steps of bisulphite removal and desulfonation, improving recovery, especially for specimens with low levels of DNA. The analytical sensitivity of the method was analysed by detection of methylated/unmethylated copies of the imprinted (and hemimethylated) gene MEST in a dilution series of plasma DNA. The method is based on an accelerated deamination step and magnetic silica purification of DNA in combination with a first round of PCR amplifying methylated and umethylated MEST. This procedure allows low levels of DNA to be easily and reliably analysed, a prerequisite for the clinical usefulness of cell-free methylated DNA detection in plasma.