title: Novel method for high-throughput colony PCR screening in nanoliter-reactors.
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.
authors.affiliation: ETH Zurich, Institute of Process Engineering, BioProcess Laboratory (BPL), Zurich, Switzerland.
authors.forename: Marcel
authors.initials: M
authors.lastname: Walser
authors.forename: Rene
authors.initials: R
authors.lastname: Pellaux
authors.forename: Andreas
authors.initials: A
authors.lastname: Meyer
authors.forename: Matthias
authors.initials: M
authors.lastname: Bechtold
authors.forename: Herve
authors.initials: H
authors.lastname: Vanderschuren
authors.forename: Richard
authors.initials: R
authors.lastname: Reinhardt
authors.forename: Joseph
authors.initials: J
authors.lastname: Magyar
authors.forename: Sven
authors.initials: S
authors.lastname: Panke
authors.forename: Martin
authors.initials: M
authors.lastname: Held
mesh_terms: D000464:Alginates / Q000737:chemistry; D001483:Base Sequence; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D002365:Manihot / Q000235:genetics; D018895:Microsatellite Repeats; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D000464:Alginates