Digital-Droplet PCR Gets Another ‘D’
Posted 2nd November 2018 by Jane Williams
Triple-d PCR enhances digital PCR sensitivity and precision for liquid biopsies
Digital-droplet PCR (ddPCR) has been implemented in diverse fields such as cancer biomarkers, viral load detection, prenatal screening, organ donor rejection, or library assessment for next generation sequencing. Detection of emerging resistance or minimal residual disease via ddPCR in liquid biopsies is also growing rapidly.
However, while interest in digital-droplet PCR technology (ddPCR) for circulating-DNA analysis is burgeoning, the paucity of starting DNA material often acts as a ‘glass ceiling’ in the molecular analysis of liquid biopsies. Irrespective how sensitive ddPCR-techniques are devised, the technology is compromised by sub-sampling errors and the number of clinical targets that can be analysed is restricted by limited number of input DNA molecules.
We now present triple-d PCR, a novel approach to this universal problem that enhances the information obtained from clinical samples with little change in established digital-PCR protocols. We show that, by appropriate denaturation of a DNA sample prior to ddPCR the information obtained is doubled, doubling also the number of possible assays and improving ddPCR precision and sensitivity.
As of its inception, digital PCR amplifies individual double stranded DNA molecules in distinct reaction compartments, then obtains signal readout from each compartment to reveal and quantify DNA targets. Yet the information contained in double stranded DNA is redundant, as each mutated base appears in both sense and antisense strands of the original molecule.
We therefore hypothesised that by applying complete denaturation of double stranded DNA just prior to droplet formation in ddPCR would double the number of positive droplets obtained from a given DNA amount, thus enhancing ddPCR analysis of clinical samples (Figure 1). Indeed, amplification using just a sense or anti-sense DNA strand in a droplet is likely to produce the same number of positive droplets as the corresponding double stranded molecule, provided the two strands are of equal size to begin with (blunt-ended DNA).
If, on the other hand, DNA is randomly fragmented with unequal strands, as in circulating DNA, then placing each strand in a separate droplet may not double the ddPCR signal, since the shorter strand may not bind the primers. We demonstrate that a DNA end-repair step performed just prior to denaturation and droplet formation restores the ability to double the ddPCR signal. We present a single-tube protocol to enable this process.
Our data shows that this simple adaptation of digital-PCR, denaturation-enhanced droplet digital PCR (dddPCR or triple-d PCR), enables extraction of more information from small input clinical samples without substantial change in existing digital PCR protocols. It should be applicable to all ddPCR platforms for mutation detection and, potentially, for gene copy-number analysis.
In view of expanding interest in using ddPCR for liquid biopsy of cancer, prenatal diagnosis or organ donor rejection, we anticipate that triple-d PCR will be widely applied in the field.
Dr Mike Makrigiorgos is a Professor of Radiation Oncology at Harvard Medical School and Director of the Medical Physics & Biophysics division at Dana Farber Cancer Institute and Brigham and Women’s Hospitals, Harvard Medical School.
Mike is presenting at the 4BIO Summit: Europe ‘Novel digital PCR and mutation enrichment technologies for the analysis of clinically relevant DNA alterations in liquid biopsies’. View the agenda to see who else is speaking.