Introduction*

Digital PCR is a new approach to nucleic acid detection and quantification, which is a different method of absolute quantification and rare allele detection relative to conventional qPCR, because it directly counts the number of target molecules rather than relying on reference standards or endogenous controls.  Below is a comparison of the different types of PCR, to help understand how it differs from Real-Time PCR.

Real Time PCR vs Traditional PCR vs Digital PCR at a Glance*

Digital PCR Real-Time PCR Traditional PCR
Overview Measures the fraction of negative replicates to determine absolute copies. Measures PCR amplification as it occurs. Measures the amount of accumulated PCR product at the end of the PCR cycles.
Quantitative? Yes, the fraction of negative PCR reactions is fit to a Poisson statistical algorithm. Yes, because data is collected during the exponential growth (log) phase of PCR when the quantity of the PCR product is directly proportional to the amount of template nucleic acid. No, though comparing the intensity of the amplified band on a gel to standards of a known concentration can give you ‘semi-quantitative’ results.
Applications
  • Absolute Quantification of Viral Load
  • Absolute Quantification of Nucleic Acid Standards
  • Absolute Quantification of Next-Gen Sequencing Libraries
  • Rare Allele Detection
  • Absolute quantification of gene expression
  • Enrichment and Separation of Mixtures
  • Quantitation of Gene Expression
  • Microarray Verification
  • Quality Control and Assay Validation
  • Pathogen detection
  • SNP Genotyping
  • Copy Number Variation
  • MicroRNA Analysis
  • Viral Quantitation
  • siRNA/RNAi experiments
Amplification of DNA for:

  • Sequencing
  • Genotyping
  • Cloning
Summary Advantages of Digital PCR:

  • No need to rely on references or standards Desired precision can be achieved by increasing total number of PCR replicates
  • Highly tolerant to inhibitors
  • Capable of analyzing complex mixtures
  • Unlike traditional qPCR, digital PCR provides a linear response to the number of copies present to allow for small fold change differences to be detected
Adavantages of Real-Time PCR:

  • Increased dynamic range of detection
  • No post-PCR processing
  • Detection is capable down to a 2-fold change
  • Collects data in the exponential growth phase of PCR
  • An increase in reporter fluorescent signal is directly proportional to the number of amplicons generated
  • The cleaved probe provides a permanent record amplification of an amplicon
Disadvantages of Traditional PCR:

  • Poor Precision
  • Low sensitivity
  • Short dynamic range < 2 logs
  • Low resolution
  • Non-Automated
  • Size-based discrimination only
  • Results are not expressed as numbers
  • Ethidium bromide for staining is not very quantitative
  • Post-PCR processing

*Taken from Lifetechnologies.com

Digital PCR works by partitioning a sample into many individual real-time PCR reactions; some portion of these reactions contain the target molecule (positive) while others do not (negative). Following PCR analysis, the fraction of negative answers is used to generate an absolute answer for the exact number of target molecules in the sample, without reference to standards or endogenous controls.

Caveats

There are some implications to this absolute count that should be understood prior to undertaking a digital experiment.

  1. Samples must be diluted to reach the digital range.  Since we are now counting molecules in a small, dilute sample, instead of looking at a concentrated sample in a large volume, the mixing of your sample prior to making a dilution is critical. Each time you dilute a sample, you take a specific number of molecules from a tube and move them to another tube.  No matter how well the sample is mixed, a different number of molecules will be removed from the tube each time you pipette from it.  Good mixing will even this out, but they will never be exactly the same.  This difference increases with each dilution step that you make.  Because of this, you may want to consider running more than one sample from different dilutions of the same stock.
  2. The results returned measure the number of copies per µl in the solution that was put onto the chip.  For this answer to have any meaning, you must have some idea of what you put on the chip.  If you want to know copies per cell, then you will need to either count the cells before you start, or have some way of measuring the number of chromosomes in your sample, i.e. use a gene like RNAse P or Tert that has only one copy per chromosome in normal cells.

Digital PCR is an exciting new technology, but it requires careful thought and planning to answer particular questions.  Because of this, consultation is required prior to starting an experiment.  Please contact us to make an appointment.