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.

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Video: Introduction to Digital PCR

Digital PCR Using the OpenArray® Real-Time PCR System

The OpenArray® Real-Time PCR System enables digital PCR experiments at a scale previously unattainable - in a single day, one user can generate >36,000 digital PCR data points on the OpenArray® Real-Time PCR System, 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 144 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
  • Wide dynamic range—with as few as 64 data points per replicate group, a dilution series can easily be loaded into the TaqMan® OpenArray® 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 to help you design your digital PCR experiment, eliminating optimization time and minimizing sample usage

Figure 1: Digital PCR uses the ratio of positive (black) to negative (white) PCR reactions to count the number of target molecules.

Digital PCR Experimental Workflow

Isolate and purify genomic DNA.

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MagMAX™ DNA Multi-Sample Kit
MagMAX™ DNA Multi-Sample Kits are designed for rapid purification of high-quality genomic DNA from a variety of sample types including blood and blood cards, tissue, mouse tails, buccal swabs, buffy coats, and cultured cells. These purification kits use MagMAX™ magnetic bead-based nucleic acid isolation technology to produce high yields of purified DNA, free from inhibitors that may affect downstream reactions such as genotyping assays, CE and next-generation sequencing, and high resolution melting analysis. MagMAX™ kits are available in both tube format (for manual processing) and in 96-well plate format (compatible with automated processing).

dPCR for Absolute Quantitation

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

Applications
  • Absolute Quantification of Viral Load
  • Absolute Quantification of Nucleic Acid Standards
  • Absolute Quantification of Next-Gen Sequencing Libraries
  • Rare Allele Detection
  • Low-Fold Copy Number Discrimination
  • Enrichment and Separation of Mixtures
 Advantages of Digital PCR:
  • 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
  • Unlike traditional qPCR, digital PCR provides a linear response to the number of copies present to to help enable analysis of small fold-change differences

Literature

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For research use only. Not intended for any animal or human therapeutic or diagnostic use.