Abstract
The standard-curve based simplex quantitative polymerase chain reaction (qPCR) has been the gold standard for DNA target quantification for more than a decade. The large and growing number of individual analyses needed to test for genetically modified organisms (GMOs) is reducing the cost-effectiveness of qPCR. Droplet digital PCR (ddPCR) enables absolute quantification without standard curves, avoids the amplification efficiency bias observed with qPCR, allows more accurate estimations at low target copy numbers and, in combination with multiplexing, significantly improves cost efficiency. Here we describe two protocols for multiplex quantification of GM maize events: (1) nondiscriminating, with multiplex quantification of targets as a group (12 GM maize lines) and (2) discriminating, with multiplex quantification of individual targets (events). The first enables the quantification of twelve European Union authorized GM maize events as a group with only two assays, but does not permit determination of the individual events present. The second protocol enables the quantification of four individual targets (three GM events and one endogene) in a single reaction. Both protocols can be modified for quantification of any other DNA target.
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Acknowledgment
This work was supported by the European Union under grant agreement no. 613908, (project DECATHLON). Complementary financial support was given by the Norwegian Research Council and the Slovenian Research Agency (contract numbers P4-0165 and 1000-15-0105). Consumables for the research resulting in the MEQ protocol were kindly provided by Bio-Rad.
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Dobnik, D. et al. (2018). Multiplex Droplet Digital PCR Protocols for Quantification of GM Maize Events. In: Karlin-Neumann, G., Bizouarn, F. (eds) Digital PCR. Methods in Molecular Biology, vol 1768. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7778-9_5
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DOI: https://doi.org/10.1007/978-1-4939-7778-9_5
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