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Novel method for high-throughput colony PCR screening in nanoliter-reactors

Walser, Marcel, Pellaux, Rene, Meyer, Andreas, Bechtold, Matthias, Vanderschuren, Herve, Reinhardt, Richard, Magyar, Joseph, Panke, Sven, Held, Martin
Nucleic acids research 2009 v.37 no.8 pp. e57
DNA, alginates, cassava, clones, fluorescence, genetic polymorphism, high-throughput nucleotide sequencing, microsatellite repeats, polymerase chain reaction, screening
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.