What is Digital PCR?

Digital PCR is a different approach to nucleic acid detection and quantification. It offers a different method for absolute quantification and rare allele detection relative to conventional real-time quantitative PCR. 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 reactions is used to generate an absolute count of the number of target molecules in the sample, without reference to standards or endogenous controls.

Applications

  • Rare allele detection
  • Absolute quantification of gene expression
  • Absolute quantification of viral load
  • Absolute quantification of nucleic acid standards
  • Absolute quantification of next-generation sequencing libraries

ADvantages

  • No need to rely on references or standards
  • Desired precision can be achieved by increasing the number of PCR replicates
  • Highly tolerant to inhibitors
  • Capable of analyzing complex mixtures
  • Provides a linear response to the number of copies present to to help enable analysis of small fold-change differences

Platforms

The GRCF offers two different digital PCR platforms. Neither platform is droplet based. Both involve partitioning the sample into individual partitions, and can be used with a variety of assays, including Taqman and PrimeTime Assays.

Constellation dPCR system

This system injects the sample into a nanoplate, which has individually sealed partitions of fixed sized. The nanoplates are formatted in a standard 96 well format, making them easily amenable to multi-channel pipetting and faster, familiar workflows. Partitioning, cycling and reading of the samples all occur on the same instrument in a very streamlined manner. Multiplexing of up to 5 assays in the same sample is possible, if all are within the digital range at the same dilution.

The nanoplates come in two different formats: 24 well, which has 36,000 partitions and 96 well, which has 8,000 partitions. The 24 well plates are ideal for looking at rare events, the 96 well plates are great for precisely counting more frequent events.

 Full platesPartial plate charge
24 well plates$11/well$11/well + $3/empty well
96 well plates$8.50/well$8.50/well + $1.50/empty well

*Please note that pricing is per well. Samples run in duplicate or triplicate will be charged for each well used.

Other possible fees:

New assay condition testing: $75/condition

Serum sample preparation: $1/sample


QuantStudio 3D

LifeTechnologies QuantStudio 3D system. This system spreads the sample onto chips, partitioning the sample into about 18,000 individual wells. This system is best for very small projects that need a large number of partitions.

Pricing:

$26/chip**

**ThermoFisher recommends running 2 chips per sample.



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

Overview

Digital PCR

Measures the fraction of negative replicates to determine absolute copies.

Real-Time PCR

Measures PCR amplification as it occurs.

Traditional PCR

Measures the amount of accumulated PCR product at the end of the PCR cycles.

Quantitative?

Digital PCR

Yes, the fraction of negative PCR reactions is fit to a Poisson statistical algorithm.

Real-Time PCR

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.

Traditional PCR

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

Digital PCR

  • 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

Real-Time PCR

  • 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

Traditional PCR

Amplification of DNA for:

  • Sequencing
  • Genotyping
  • Cloning

Summary

Digital PCR

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

Real-Time PCR

Advantages 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

Traditional PCR

Disadvantages of Traditional PCR:

  • Poor PrecisionLow 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.