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Applied and Environmental Microbiology, February 2001, p. 880-887, Vol. 67, No. 2
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.2.880-887.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Evaluation of PCR-Generated Chimeras, Mutations, and Heteroduplexes with 16S rRNA Gene-Based Cloning

Xiaoyun Qiu,1,2 Liyou Wu,1,2 Heshu Huang,1 Patrick E. McDonel,1 Anthony V. Palumbo,1 James M. Tiedje,2 and Jizhong Zhou1,2,*

Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 38831,1 and Center for Microbial Ecology, Michigan State University, East Lansing, Michigan 488242

Received 8 June 2000/Accepted 30 October 2000

To evaluate PCR-generated artifacts (i.e., chimeras, mutations, and heteroduplexes) with the 16S ribosomal DNA (rDNA)-based cloning approach, a model community of four species was constructed from alpha, beta, and gamma subdivisions of the division Proteobacteria as well as gram-positive bacterium, all of which could be distinguished by HhaI restriction digestion patterns. The overall PCR artifacts were significantly different among the three Taq DNA polymerases examined: 20% for Z-Taq, with the highest processitivity; 15% for LA-Taq, with the highest fidelity and intermediate processitivity; and 7% for the conventionally used DNA polymerase, AmpliTaq. In contrast to the theoretical prediction, the frequency of chimeras for both Z-Taq (8.7%) and LA-Taq (6.2%) was higher than that for AmpliTaq (2.5%). The frequencies of chimeras and of heteroduplexes for Z-Taq were almost three times higher than those of AmpliTaq. The total PCR artifacts increased as PCR cycles and template concentrations increased and decreased as elongation time increased. Generally the frequency of chimeras was lower than that of mutations but higher than that of heteroduplexes. The total PCR artifacts as well as the frequency of heteroduplexes increased as the species diversity increased. PCR artifacts were significantly reduced by using AmpliTaq and fewer PCR cycles (fewer than 20 cycles), and the heteroduplexes could be effectively removed from PCR products prior to cloning by polyacrylamide gel purification or T7 endonuclease I digestion. Based upon these results, an optimal approach is proposed to minimize PCR artifacts in 16S rDNA-based microbial community studies.

* Corresponding author. Mailing address: Environmental Sciences Division, Oak Ridge National Laboratory, P. O. Box 2008, Oak Ridge, TN 37831-6038. Phone: (865) 576-7544. Fax: (865) 576-8646. E-mail: zhouj{at}ornl.gov.

Applied and Environmental Microbiology, February 2001, p. 880-887, Vol. 67, No. 2
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.2.880-887.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.