Digital PCR

Digital PCR (dPCR) is a quantitative PCR method that provides a sensitive and reproducible way of measuring the amount of DNA or RNA present in a sample. This method is similar to qPCR in the reaction assembly components and amplification reaction, but differs in the way the sample target is measured.  Digital PCR is a simple and reproducible method that does not rely on a calibration curve for sample target quantification.  No reference standards or endogenous controls are needed.  For dPCR, the initial sample mix is partitioned into a large number of individual wells prior to the amplification step, resulting in either 1 or 0 targets being present in each well.  Following PCR amplification, the number of positive vs negative reactions is determined and the absolute quantification of target calculated using Poisson statistics.

The Genomics Core Facility is equipped with QuantStudio 3D, a chip-based dPCR system developed by ThermoFisher. The QuantStudio 3D Digital PCR Chip enables a simple and streamlined workflow. Unlike other methods that require multiple transfer steps and upfront sample digestion, the QuantStudio 3D Digital PCR Chip is simply loaded and sealed—ready for PCR and analysis. With the ability to thermal cycle up to 24 chips at one time and taking less than 30 seconds to read each chip, the QuantStudio 3D Digital PCR System enables a simple, streamlined workflow to perform digital PCR. Data analysis is performed in ThermoFisher cloud computing environment using applications specifically developed for every type of dPCR project.

Main applications of Digital PCR include:

  1. Absolute quantification of DNA, Viral load and low-level pathogen detection/quantification
  2. Sensitive Mutation Detection  using TaqMan Liquid Biopsy dPCR Assays
  3. Copy Number Variation
  4. Next Generation Sequencing (NGS) Library Quantification
  5. Absolute quantification of Standards and References
  6. Differential Gene Expression ( < 2-fold change using housekeeping reference gene as a standard).