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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,
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.

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

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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