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Applied and Environmental Microbiology, January 2001, p. 469-472, Vol. 67, No. 1
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.1.469-472.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Microvariation Artifacts Introduced by PCR and Cloning of Closely Related 16S rRNA Gene Sequences†

Arjen G. C. L. Speksnijder,1,2,* George A. Kowalchuk,3 Sander De Jong,2 Elizabeth Kline,4 John R. Stephen,4,Dagger and Hendrikus J. Laanbroek2

Department of Zoology, Natural History Museum, South Kensington, London SW7 5BD, United Kingdom1; Department of Plant-Microorganism Interactions, Centre for Terrestrial Ecology, Netherlands Institute of Ecology, 6666 ZG Heteren,3 and Department of Microbial Ecology, Centre for Limnology, Netherlands Institute of Ecology, 3136 AC Nieuwersluis,2 The Netherlands; and Center for Environmental Biotechnology, University of Tennessee, Knoxville, Tennessee 37932-25754

Received 3 August 2000/Accepted 6 October 2000

A defined template mixture of seven closely related 16S-rDNA clones was used in a PCR-cloning experiment to assess and track sources of artifactual sequence variation in 16S rDNA clone libraries. At least 14% of the recovered clones contained aberrations. Artifact sources were polymerase errors, a mutational hot spot, and cloning of heteroduplexes and chimeras. These data may partially explain the high degree of microheterogeneity typical of sequence clusters detected in environmental clone libraries.

* Corresponding author. Present address: Dept. of Molecular and Cell Biology, IMS Building, Foresterhill, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom. Phone: 44 1224 273149. Fax: 44 1224 273144. E-mail: a.speksnijder{at}abdn.ac.uk.

dagger Publication 2679 of the NIOO Centre for Limnology, Nieuwersluis, The Netherlands.

Dagger Present address: Crop and Weed Science, Horticulture Research International, Wellesbourne, UK CV35 9EF.

Applied and Environmental Microbiology, January 2001, p. 469-472, Vol. 67, No. 1
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.1.469-472.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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