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dPCR – the digital polymerase chain reaction

Abstract

Quantitative real-time PCR (qPCR) is based on the assumption that the amplification of DNA target molecules is exponential. DNA targets may be quantified by comparing the number of amplification cycles required to achieve a predetermined signal threshold to that obtained for a calibrant. However, many factors complicate this calculation, creating uncertainties and inaccuracies. Digital PCR (dPCR) is a modification of the qPCR method that can be employed to quantify precisely defined nucleic acid targets. The technique is based on the concept of limiting dilutions, which involves the partitioning of a PCR reaction into multiple sub-reactions such that each sub-reaction either contains none or one or more DNA targets. Following thermal cycling, reactions are classified as either positive (target detected) or negative (no target detected), hence providing the basis for a digital output format. By determining the proportion of empty partitions, Poissonian statistics can be applied and the initial number of target molecules present can be estimated.

Graphical abstract: dPCR – the digital polymerase chain reaction

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Publication details

The article was received on 23 Jun 2017 and first published on 07 Jul 2017


Article type: AMC Technical Brief
DOI: 10.1039/C7AY90093G
Citation: Anal. Methods, 2017, Advance Article
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    dPCR – the digital polymerase chain reaction

    Analytical Methods Committee AMCTB No. 79, Anal. Methods, 2017, Advance Article , DOI: 10.1039/C7AY90093G

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