Identification of rare levels of methylated tumor DNA fragments using an optimized bias based pre-amplification-digital droplet PCR (OBBPA-ddPCR)

Background: The analysis of aberrant DNA methylation is used for the diagnosis of cancer as significant changes in the gene methylation pattern are often detected during early carcinogenesis. In this study, we evaluated the performance of a two-step method that combines pre-amplification with ddPCR technique. Results: By using ddPCR, the dependence of amplification efficiency for methylated and unmethylated DNA fragments on the relevant MgCl ₂ concentration and the annealing temperature was established in addition to the primer design. We found that the efficiency can be adjusted toward methylated sequences by using primers covering one to four CpG sites under appropriately selected MgCl ₂ concentration and annealing temperature. Applying a PCR bias between 85% and 95%, five copies of methylated tumor DNA fragments were detected against a background of 700,000 copies of unmethylated DNA fragments with a high signal-to-noise ratio. The analysis of serum samples from patients with prostate cancer showed a significantly improved performance of the new method in comparison with the MS-HRM technique, ddPCR alone, or ddPCR in combination with an unbiased pre-amplification using methylation-independent primers. Conclusions: We define this method as an optimized bias-based pre-amplification-digital droplet PCR (OBBPA-ddPCR) technique. This novel method is recommended for the early detection of cancer-specific DNA methylation biomarkers in the form of a liquid biopsy.