Digital PCR

a new approach to nucleic
acid detection and quantification

 


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Video: Introduction to Digital PCR >

Digital PCR is a new 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.
See how digital PCR and real-time PCR compare >

Digital PCR

QuantStudio™ 3D Digital PCR System

Digital PCR for Absolute Quantitation

Digital PCR is quantitative PCR method that can be used for absolute target quantitation.

ApplicationsAdvantages of Digital PCR
  • 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
  • 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

Digital PCR on QuantStudio® 12K Flex Real-Time PCR System

The QuantStudio® 12K Flex Real-Time PCR System enables digital PCR experiments at a scale previously unattainable - in a single day, one user can generate >49,000 digital PCR data points, without the use of robotics. Other features of the system include:

  • Accuracy and sensitivity—detect and count individual molecules to quantify viral load, gDNA or cDNA targets, plasmids, or next-generation sequencing libraries.
  • Speed—produce up to 192 digital answers in a 3 hour run.
  • Flexibility—use your existing real-time PCR assays and test from one to 48 assays/sample dilutions per plate, with a combination of 4 plates.
  • Wide dynamic range—with as few as 64 data points per replicate group, a dilution series can easily be loaded into the QuantStudio® Digital PCR Plate, expanding the range of sample concentrations which can be analyzed to produce a digital answer.
  • Intuitive and economical to use—software includes a Poisson calculator, and measures values in real-time, to help you design your digital PCR experiment, eliminating optimization time and minimizing sample usage.

Digital PCR Workflow

1 Select assays or panels
step 1

Select assays and QuantStudio™ Digital PCR plates. Order products.

2 Add samples & master mix
step 2

Mix assays, samples, and master mix in 384-well sample plates.

3 Load samples

step 3

Load sample mixes onto QuantStudio™ Digital PCR plate(s) with the QuantStudio™ 12K Flex AccuFill™ System.

4 Apply lid & fill
step 4

The QuantStudio™ Digital PCR plate is encased in an alloy bottom for easy handling; simply apply the adhesive lid and fill case with immersion fluid.

5 Cycle, image, analyze data
step 5

Easily run up to 4 QuantStudio™ Digital PCR plates for any application on the QuantStudio™ 12K Flex instrument.

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Literature

Digital PCR on the QuantStudio™ 12K Flex Real-Time PCR System

Product Bulletin: OpenArray® Real-Time PCR System

User Guide: OpenArray® Real-Time PCR System, Digital PCR Experiment

App Note: Single-cell profiling on the OpenArray® Real-Time PCR System

App Note: Digital PCR for Virology Research-Beyond Ct

Publications

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Regulatory Status: For Research Use Only. Not for human or animal therapeutic or diagnostic use.

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