{"id":6999,"date":"2015-03-16T13:27:26","date_gmt":"2015-03-16T13:27:26","guid":{"rendered":"http:\/\/admin.acceleratingscience.com\/?p=6999"},"modified":"2022-06-02T22:17:22","modified_gmt":"2022-06-02T22:17:22","slug":"what-is-digital-pcr","status":"publish","type":"post","link":"https:\/\/www.thermofisher.com\/blog\/behindthebench\/what-is-digital-pcr\/","title":{"rendered":"What Is Digital PCR?"},"content":{"rendered":"

Digital PCR<\/strong> \u00a0is a specialized approach to nucleic acid detection and quantification that estimates absolute numbers of molecules through statistical methods. Digital PCR (dPCR) <\/strong>uses the same fundamental chemistry as qPCR, but unlike qPCR data, dPCR data are collected at the endpoint of the reaction mix. Before amplification, a bulk PCR reaction made up of nucleic acid, primers, probes, and master mix is digitized into many thousands of nanoliter-sized microreactions<\/a>. As this digitization process distributes the PCR mix across so many microreactions, each microreaction will effectively either contain one, zero, or just a handful of the target nucleic acid molecules. The isolated microreactions are then amplified, and data are collected from each microreaction at the end of the thermal cycling process. Microreactions that do not contain the target will not show post-amplification fluorescence, while those that do contain the target will show post-amplification fluorescence.<\/p>\n

In effect, the original reaction is turned into many binary reactions. After counting the positive microreactions, simple statistics can be used to then determine the “absolute” quantity of the target molecule rather than a quantity estimated by comparing to a standard of known concentration. \u00a0This technology offers an alternative to qPCR for absolute quantification and rare allele detection which does not rely on the number of amplification cycles to determine the initial amount of template nucleic acid in each sample.<\/p>\n

To date, qPCR has been a powerful and sensitive gene analysis technique used for a broad range of applications. As the name suggests, qPCR measures PCR amplification as it occurs, unlike traditional PCR, which collects results after the reaction is complete, making it impossible to determine the starting concentration of nucleic acid. qPCR’s entry into the market revolutionized PCR-based quantitation of DNA and RNA.<\/p>\n

Digital PCR vs. Real-Time PCR vs. Traditional PCR<\/h2>\n

To date, qPCR has been a powerful and sensitive gene analysis techniques used for a broad range of applications. As the name suggests, qPCR measures PCR amplification as it occurs, unlike traditional PCR, which collects results after the reaction is complete, making it impossible to determine the starting concentration of nucleic acid. qPCR\u2019s entry into the market revolutionized PCR-based quantitation of DNA and RNA.<\/p>\n

Digital PCR is a newer approach to nucleic acid detection and quantification that estimates absolute numbers of molecules through statistical methods. This technology offers an alternative to qPCR for absolute quantification and rare allele detection rather than relying on the number of amplification cycles to determine the initial amount of template nucleic acid in each sample.<\/p>\n\n\n\n\n\n\n\n\n
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Digital PCR vs. Real-Time qPCR vs. Traditional PCR at a Glance<\/strong><\/a><\/p>\n<\/td>\n<\/tr>\n

<\/td>\n\n

Digital PCR<\/strong><\/span><\/p>\n<\/td>\n

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Real-Time qPCR<\/strong><\/span><\/p>\n<\/td>\n

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Traditional PCR<\/strong><\/span><\/p>\n<\/td>\n<\/tr>\n

Overview<\/strong><\/span><\/td>\n\n

Measures the fraction of negative microreactions to determine absolute copies.<\/p>\n<\/td>\n

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Measures PCR amplification as it occurs in a bulk reaction mix.<\/p>\n<\/td>\n

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Measures the amount of accumulated PCR product at the end of the PCR cycles.<\/p>\n<\/td>\n<\/tr>\n

Quantitative?<\/strong><\/span><\/td>\n\n

Yes \u2013 The fraction of negative microreactions is fit to a Poisson statistical algorithm.<\/p>\n<\/td>\n

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Yes \u2013 Data is collected during the exponential growth phase of PCR when the quantity of the PCR product is directly proportional to the amount of template nucleic acid.<\/p>\n<\/td>\n

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No \u2013 However, comparing the intensity of the amplified band on a gel to standards of a known concentration can give you semi-quantitative results.<\/p>\n<\/td>\n<\/tr>\n

Applications<\/strong><\/span><\/td>\n\n
    \n
  • Absolute quantification of viral load<\/li>\n
  • Absolute quantification of nucleic acid standards<\/li>\n
  • Absolute quantification of next-gen sequencing Libraries<\/li>\n
  • Rare allele detection<\/li>\n
  • Absolute quantification of gene expression<\/li>\n
  • Analysis of multiple targets on a single molecule through multiplexing<\/li>\n<\/ul>\n<\/td>\n
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    \n
  • Quantitation of gene expression<\/li>\n
  • Microarray verification<\/li>\n
  • Quality control and assay validation<\/li>\n
  • Pathogen detection<\/li>\n
  • SNP genotyping<\/li>\n
  • Copy number variation<\/li>\n
  • MicroRNA analysis<\/li>\n
  • Viral quantitation<\/li>\n
  • \u2022 siRNA\/RNAi experiments<\/li>\n<\/ul>\n<\/td>\n
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    \n
  • Amplification of DNA for:<\/li>\n
  • \u2022 Sequencing<\/li>\n
  • \u2022 Genotyping<\/li>\n
  • \u2022 Cloning<\/li>\n<\/ul>\n<\/td>\n<\/tr>\n
Advantages<\/strong><\/span><\/td>\n\n
    \n
  • No need to rely on references or standards<\/li>\n
  • Desired precision can be achieved by increasing total number of PCR replicates<\/li>\n
  • More tolerant to some PCR inhibitors<\/li>\n
  • Capable of analyzing complex mixtures<\/li>\n
  • Provides a linear response to the number of copies present to allow for small fold change differences to be detected<\/li>\n<\/ul>\n<\/td>\n
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    \n
  • Increased dynamic range of detection<\/li>\n
  • No post-PCR processing<\/li>\n
  • Detection is capable down to a 2-fold change<\/li>\n
  • Collects data in the exponential growth phase of PCR<\/li>\n
  • An increase in reporter fluorescent signal is directly proportional to the number of amplicons generated<\/li>\n
  • The cleaved probe provides a permanent record amplification of an amplicon<\/li>\n<\/ul>\n<\/td>\n
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