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

Alginates/chemistry; Base Sequence; Conserved Sequence; Escherichia coli/genetics; Gene Library; Manihot/genetics; Microsatellite Repeats; Polymerase Chain Reaction/methods; Polymorphism, Genetic; Sequence Analysis, DNA/methods

Abstract :

[en] 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.

Disciplines :

Biochemistry, biophysics & molecular biology

Author, co-author :

Walser, Marcel

Pellaux, Rene

Meyer, Andreas

Bechtold, Matthias

Vanderschuren, Hervé ; Université de Liège - ULiège > Sciences agronomiques > Biologie végétale

Reinhardt, Richard

Magyar, Joseph

Panke, Sven

Held, Martin

Language :

English

Title :

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

Publication date :

2009

Journal title :

Nucleic Acids Research

ISSN :

0305-1048

eISSN :

1362-4962

Publisher :

Oxford University Press, United Kingdom

Volume :

Issue :

Pages :

e57

Peer reviewed :

Peer Reviewed verified by ORBi

Available on ORBi :

since 06 October 2014

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