Single-molecule resolution mutation detection using single-color droplet digital PCR from un-amplified circulating tumor DNA
Our novel single color digital droplet PCR (ddPCR) molecular assay can detect and quantifying cancer mutations directly from poor quality and limited quantity DNA samples such as un-amplified circulating tumor DNA (ctDNA) collected from the plasma of cancer patients.
The single-color mutation quantitation relies on double stranded DNA binding dye and two allele specific DNA primer sets in order to produce an absolute count of mutant and wild type DNA molecules present in the original sample. This simple and low-cost assay can be rapidly configured for nearly any cancer mutation of interest, making it a potentially invaluable tool for precision medicine and customized longitudinal monitoring of cancer patients.
From an input of 1 nanogram of un-amplified DNA, equivalent to ~300 genomic molecules, we have demonstrated a sensitivity of 1% molecular limit of detection (LOD) or 3 mutant DNA molecules per assay reaction well. This LOD can be improved to resolve a 0.1% LOD by increasing the number of replicates or genome equivalents assayed per given sample. We have validated this technology for cancer patients and are using this technology to longitudinally monitor these patients over the course of their individual treatments.