Novel method for high-throughput colony PCR screening in nanoliter-reactors |
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| Authors: | Marcel Walser Rene Pellaux Andreas Meyer Matthias Bechtold Herve Vanderschuren Richard Reinhardt Joseph Magyar Sven Panke Martin Held |
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| Affiliation: | 1.ETH Zurich, Institute of Process Engineering, BioProcess Laboratory (BPL), 2.ETH Zurich, Institute of Plant Science, Plant Biotechnology, Zurich, Switzerland, 3.Max Planck Institute for Molecular Genetics, Analytics & Computing, Berlin, Germany and 4.Harlan Laboratories Ltd., Environmental Safety and Metabolism, Itingen, Switzerland |
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| Abstract: | We introduce a technology for the rapid identification and sequencing of conserved DNA elements employing a novel suspension array based on nanoliter (nl)-reactors made from alginate. The reactors have a volume of 35 nl and serve as reaction compartments during monoseptic growth of microbial library clones, colony lysis, thermocycling and screening for sequence motifs via semi-quantitative fluorescence analyses. nl-Reactors were kept in suspension during all high-throughput steps which allowed performing the protocol in a highly space-effective fashion and at negligible expenses of consumables and reagents. As a first application, 11 high-quality microsatellites for polymorphism studies in cassava were isolated and sequenced out of a library of 20 000 clones in 2 days. The technology is widely scalable and we envision that throughputs for nl-reactor based screenings can be increased up to 100 000 and more samples per day thereby efficiently complementing protocols based on established deep-sequencing technologies. |
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