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Issue 13, 2011
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Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR

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Quantitative polymerase chain reactions (qPCR) based on real-time PCR constitute a powerful and sensitive method for the analysis of nucleic acids. However, in qPCR, the ability to multiplex targets using differently colored fluorescent probes is typically limited to 4-fold by the spectral overlap of the fluorophores. Furthermore, multiplexing qPCR assays requires expensive instrumentation and most often lengthy assay development cycles. Digital PCR (dPCR), which is based on the amplification of single target DNA molecules in many separate reactions, is an attractive alternative to qPCR. Here we report a novel and easy method for multiplexing dPCR in picolitre droplets within emulsions—generated and read out in microfluidic devices—that takes advantage of both the very high numbers of reactions possible within emulsions (>106) as well as the high likelihood that the amplification of only a single target DNA molecule will initiate within each droplet. By varying the concentration of different fluorogenic probes of the same color, it is possible to identify the different probes on the basis of fluorescence intensity. Adding multiple colors increases the number of possible reactions geometrically, rather than linearly as with qPCR. Accurate and precise copy numbers of up to sixteen per cell were measured using a model system. A 5-plex assay for spinal muscular atrophy was demonstrated with just two fluorophores to simultaneously measure the copy number of two genes (SMN1 and SMN2) and to genotype a single nucleotide polymorphism (c.815A>G, SMN1). Results of a pilot study with SMA patients are presented.

Graphical abstract: Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR

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Publication details

The article was received on 13 Feb 2011, accepted on 20 Apr 2011 and first published on 17 May 2011

Article type: Paper
DOI: 10.1039/C1LC20126C
Citation: Lab Chip, 2011,11, 2167-2174

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    Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR

    Q. Zhong, S. Bhattacharya, S. Kotsopoulos, J. Olson, V. Taly, A. D. Griffiths, D. R. Link and J. W. Larson, Lab Chip, 2011, 11, 2167
    DOI: 10.1039/C1LC20126C

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