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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Bibliography

Hutchison,C.A. III. (2007) DNA sequencing: Bench to bedside and beyond. Nucleic Acids Res., 35, 6227-6237.
Lorenz,P. and Eck,J. (2005) Metagenomics and industrial applications. Nat. Rev., 3, 510-516.
Farrar,K. and Donnison,I. (2007) Construction and screening of BAC libraries made from brachypodium genomic DNA. Nat. Protoc., 2, 1661-1674.
Beja,O. (2004) To BAC or not to BAC: Marine ecogenomics. Curr. Opin. Biotechnol., 15, 187-190.
Venter,J.C., Remington,K., Heidelberg,J.F., Halpern,A.L., Rusch,D., Eisen,J.A., Wu,D., Paulsen,I., Nelson,K.E., Nelson,W. et al. (2004) Environmental genome shotgun sequencing of the Sargasso Sea. Science, 304, 66-74.
Dahlroth,S.L., Nordlund,P. and Cornvik,T. (2006) Colony filtration blotting for screening soluble expression in Escherichia coli. Nat. Protoc., 1, 253-258.
Jones,P., Watson,A., Davies,M. and Stubbings,S. (1992) Integration of image analysis and robotics into a fully automated colony picking and plate handling system. Nucleic Acids Res., 20, 4599-4606.
Kim,C.G., Fujiyama,A. and Saitou,N. (2003) Construction of a gorilla fosmid library and its PCR screening system. Genomics, 82, 571-574.
Shendure,J. and Ji,H. (2008) Next-generation DNA sequencing. Nat. Biotechnol., 26, 1135-1145.
Freeman,A., Cohen-Hadar,N., Abramow,S., Modai-Hod,R., Dror,Y. and Georgiou,G. (2004) Screening of large protein libraries by the 'cell immobilized on adsorbed bead' approach. Biotechnol. Bioeng., 86 196-200.
Dressman,D., Yan,H., Traverso,G., Kinzler,K.W. and Vogelstein,B. (2003) Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proc. Natl Acad. Sci. USA, 100, 8817-8822.
Fan,J.B. and Chee,M.S. (2006) Highly parallel genomic assays. Nature Rev., 7, 632-644.
Lee,Y.-F., Tawfik,D.S. and Griffiths,A.D. (2002) Investigating the target recognition of DNA cytosine-5 methyltransferase HhaI by library selection using in vitro compartmentalisation. Nucleic Acids Res., 30, 4937-4944.
Miller,O.J., Bernath,K., Agresti,J., Amitai,G., Kelly,B.T., Mastrobattista,E., Taly,V., Magdassi,S., Tawfik,D.S. and Griffiths,A.D. (2006) Directed evolution by in vitro compartmentalization. Nat. Methods, 3, 561-570.
Leamon,J.H., Link,D.R., Egholm,M. and Rothberg,J.M. (2006) Overview: methods and applications for droplet compartmentalization of biology. Nat. Methods, 3, 541-543.
Diehl,F., Li,M., He,Y., Kinzler,K.W., Vogelstein,B. and Dressman,D. (2006) BEAMing: Single-molecule PCR on microparticles in water-in-oil emulsions. Nat. Methods, 3, 551-559.
Margulies,M., Egholm,M., Altman,W.E., Attiya,S., Bader,J.S., Bemben,L.A., Berka,J., Braverman,M.S., Chen,Z., Dewell,S.B. et al. (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature, 437, 376-380.
Cowan,D., Meyer,Q., Stafford,W., Muyanga,S., Cameron,R. and Wittwer,P. (2005) Metagenomic gene discovery: Past, present, future. Trends Biotechnol., 23, 321-329.
Inglese,J., Johnson,R.L., Simeonov,A., Xia,M., Zheng,W., Austin,C.P. and Auld,D.S. (2007) High-throughput screening assays for the identification of chemical probes. Nat. Chem. Biol., 3, 466-479.
Mampel,J., Spirig,T., Weber,S.S., Haagensen,J.A.J., Molin,S. and Hilbi,H. (2006) Planktonic replication is essential for biofilm formation by legionella pneumophila in a complex medium under static and dynamic flow conditions. Appl. Environ. Microbiol., 72, 2885-2895.
Walser,M., Leibundgut,R., Pellaux,R., Panke,S. and Held,M. (2008) Isolation of monoclonal microcarriers colonized by fluorescent E. coli. Cytometry Part A, 73A, 788-798.
Allen,G.C., Flores-Vergara,M.A., Krasnyanski,S., Kumar,S. and Thompson,W.F. (2006) A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nat. Protoc., 1, 2320-2325.
Anderson,J.A., Churchill,G.A., Autrique,J.E., Tanksley,S.D. and Sorrells,M.E. (1993) Optimizing parental selection for genetic linkage maps. Genome, 36, 181-186.
Brandenberger,H. and Widmer,F. (1999) Immobilization of highly concentrated cell suspensions using the laminar jet breakup technique. Biotechnol Prog., 15, 366-372.
Okogbenin,E., Marin,J. and Fregene,M. (2006) An SSR-based molecular genetic map of cassava. Euphytica, 147, 433-440.
Gupta,P.K. and Varshney,R.K. (2000) The development and use of microsatellite markers for genetic analysis and plant breeding with emphasis on bread wheat. Euphytica, 113, 163-185.
Squirrell,J., Hollingsworth,P.M., Woodhead,M., Russell,J., Lowe,A.J., Gibby,M. and Powell,W. (2003) How much effort is required to isolate nuclear microsatellites from plants? Mol. Ecol., 12, 1339-1348.
Terai,G., Komori,T., Asai,K. and Kin,T. (2007) miRRim: A novel system to find conserved miRNAs with high sensitivity and specificity. RNA, 13, 2081-2090.
Ohler,U.W.E., Yekta,S., Lim,L.P., Bartel,D.P. and Burge,C.B. (2004) Patterns of flanking sequence conservation and a characteristic upstream motif for microRNA gene identification. RNA, 10, 1309-1322.