Novel method for high-throughput colony PCR screening in nanoliter-reactors.
- Walser M ETH Zurich, Institute of Process Engineering, BioProcess Laboratory (BPL), Zurich, Switzerland.
- Pellaux R
- Meyer A
- Bechtold M
- Vanderschuren H
- Reinhardt R
- Magyar J
- Panke S
- Held M
- 2009-03-14
Published in:
- Nucleic acids research. - 2009
Alginates
Base Sequence
Conserved Sequence
Escherichia coli
Gene Library
Manihot
Microsatellite Repeats
Polymerase Chain Reaction
Polymorphism, Genetic
Sequence Analysis, DNA
English
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.
- Language
-
- English
- Open access status
- gold
- Identifiers
-
- DOI 10.1093/nar/gkp160
- PMID 19282448
- Persistent URL
- https://sonar.ch/global/documents/47299
Statistics
Document views: 24
File downloads:
- Full-text: 0