Core Molecular Biology and Genomics Laboratory

Droplet Digital PCR

Droplet Digital polymerase chain reaction (ddPCR) provides high-precision, absolute quantification of nucleic acid, capable of detecting small fold changes in expression with a wide range of applications for both research and clinical diagnostic applications. ddPCR has the following benefits for nucleic acid quantification:

  • Unparalleled precision – the massive sample partitioning afforded by ddPCR enables detection of small fold changes or rare targets.
  • Less susceptible to inhibition.
  • Simplified quantification.

 

How does it work:

Samples are placed into the droplet generator which partitions each sample into 20,000 nanoliter sized droplets. The droplets are then transferred to a thermal cycler where your gene of interest is amplified. Following amplification of your target in droplets, the samples are then injected into the droplet reader. The reader measures fluorescence in each droplet using a two-color detection system which allows for duplex experiments. Droplets which contain at least one copy of your target exhibit fluorescence and is counted as a positive. The QuantaSoft TM software then fits the fraction of positive droplets to a Poisson algorithm to determine the starting concentration of the target in units of copies/uL input.

Past Work:

 Dr. R. Godoy. “I am interested in understanding the regenerative ability of dopaminergic neurons in the adult zebrafish brain. We use ddPCR to look at the transcript levels of candidate genes which may be deferentially regulated during regeneration as an initial approach to elucidate the molecular mechanisms governing this process. ddPCR makes it possible for us to analyze such data despite a limited tissue availability and a low transcript level for some of our targets, which would otherwise not be feasible when using the conventional RT-qPCR approach.”

Dr. C Boddy. “As the genes of interest are products of genes that have low transcript levels, we did ddPCR to analyze a synthetic compound’s ability to interrupt the regulon of a sigma factor.”

Dr. A. Poulain. “We used ddPCR to determine the quantity of standards for quantitative PCR, which we will use on relevant functional genes from environmental samples.”

More information:

To discuss if ddPCR is an application or solution for your project, to discuss sample specifics, fees, or for any further information, please contact Philip Pelletier.

Updated: Jan 8, 2016