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Nucleic Acids Research Advance Access published online on March 12, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp160
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© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

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

Walser Marcel1, Pellaux Rene1, Meyer Andreas1, Bechtold Matthias1, Vanderschuren Herve2, Reinhardt Richard3, Magyar Joseph4, Panke Sven1 and Held Martin1,*

1ETH Zurich, Institute of Process Engineering, BioProcess Laboratory (BPL), 2ETH Zurich, Institute of Plant Science, Plant Biotechnology, Zurich, Switzerland, 3Max Planck Institute for Molecular Genetics, Analytics & Computing, Berlin, Germany and 4Harlan Laboratories Ltd., Environmental Safety and Metabolism, Itingen, Switzerland

*To whom correspondence should be addressed. Tel: +41 44 632 4315; Fax: +41 44 632 1325; Email: martin.held{at}bsse.ethz.ch

Received December 17, 2008. Revised February 26, 2009. Accepted February 26, 2009.

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.

Author's contributions

M.W. carried out the described experiments and wrote the manuscript. M.B. contributed with thermocycler programming. H.V. reviewed the plant part of the manuscript. R.P., A.J.M., R.R., J.M. and S.P. supervised and improved critical parts of the work. M.H. was the responsible senior researcher on the project.


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