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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.