This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Qiu, X.
Right arrow Articles by Zhou, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Qiu, X.
Right arrow Articles by Zhou, J.
Agricola
Right arrow Articles by Qiu, X.
Right arrow Articles by Zhou, J.

 Previous Article  |  Next Article 

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.


This article has been cited by other articles:

  • Nikolausz, M., Chatzinotas, A., Tancsics, A., Imfeld, G., Kastner, M. (2009). Evaluation of Single-Nucleotide Primer Extension for Detection and Typing of Phylogenetic Markers Used for Investigation of Microbial Communities. Appl. Environ. Microbiol. 75: 2850-2860 [Abstract] [Full Text]  
  • Blow, M. J., Zhang, T., Woyke, T., Speller, C. F., Krivoshapkin, A., Yang, D. Y., Derevianko, A., Rubin, E. M. (2008). Identification of ancient remains through genomic sequencing. Genome Res 18: 1347-1353 [Abstract] [Full Text]  
  • Brotherton, P., Endicott, P., Sanchez, J. J., Beaumont, M., Barnett, R., Austin, J., Cooper, A. (2007). Novel high-resolution characterization of ancient DNA reveals C > U-type base modification events as the sole cause of post mortem miscoding lesions. Nucleic Acids Res 35: 5717-5728 [Abstract] [Full Text]  
  • Nakatsu, C. H. (2007). Soil Microbial Community Analysis Using Denaturing Gradient Gel Electrophoresis. Soil Sci. 71: 562-571 [Abstract] [Full Text]  
  • Hartmann, M., Widmer, F. (2006). Community Structure Analyses Are More Sensitive to Differences in Soil Bacterial Communities than Anonymous Diversity Indices. Appl. Environ. Microbiol. 72: 7804-7812 [Abstract] [Full Text]  
  • Danovaro, R., Luna, G. M., Dell'Anno, A., Pietrangeli, B. (2006). Comparison of Two Fingerprinting Techniques, Terminal Restriction Fragment Length Polymorphism and Automated Ribosomal Intergenic Spacer Analysis, for Determination of Bacterial Diversity in Aquatic Environments. Appl. Environ. Microbiol. 72: 5982-5989 [Abstract] [Full Text]  
  • Acinas, S. G., Sarma-Rupavtarm, R., Klepac-Ceraj, V., Polz, M. F. (2005). PCR-Induced Sequence Artifacts and Bias: Insights from Comparison of Two 16S rRNA Clone Libraries Constructed from the Same Sample. Appl. Environ. Microbiol. 71: 8966-8969 [Abstract] [Full Text]  
  • Hongoh, Y., Deevong, P., Inoue, T., Moriya, S., Trakulnaleamsai, S., Ohkuma, M., Vongkaluang, C., Noparatnaraporn, N., Kudo, T. (2005). Intra- and Interspecific Comparisons of Bacterial Diversity and Community Structure Support Coevolution of Gut Microbiota and Termite Host. Appl. Environ. Microbiol. 71: 6590-6599 [Abstract] [Full Text]  
  • Castaldini, M., Turrini, A., Sbrana, C., Benedetti, A., Marchionni, M., Mocali, S., Fabiani, A., Landi, S., Santomassimo, F., Pietrangeli, B., Nuti, M. P., Miclaus, N., Giovannetti, M. (2005). Impact of Bt Corn on Rhizospheric and Soil Eubacterial Communities and on Beneficial Mycorrhizal Symbiosis in Experimental Microcosms. Appl. Environ. Microbiol. 71: 6719-6729 [Abstract] [Full Text]  
  • COLE-TOBIAN, J. L., BIASOR, M., KING, C. L. (2005). HIGH COMPLEXITY OF PLASMODIUM VIVAX INFECTIONS IN PAPUA NEW GUINEAN CHILDREN. Am J Trop Med Hyg 73: 626-633 [Abstract] [Full Text]  
  • Duperron, S., Nadalig, T., Caprais, J.-C., Sibuet, M., Fiala-Medioni, A., Amann, R., Dubilier, N. (2005). Dual Symbiosis in a Bathymodiolus sp. Mussel from a Methane Seep on the Gabon Continental Margin (Southeast Atlantic): 16S rRNA Phylogeny and Distribution of the Symbionts in Gills. Appl. Environ. Microbiol. 71: 1694-1700 [Abstract] [Full Text]  
  • Hill, J. E., Hemmingsen, S. M., Goldade, B. G., Dumonceaux, T. J., Klassen, J., Zijlstra, R. T., Goh, S. H., Van Kessel, A. G. (2005). Comparison of Ileum Microflora of Pigs Fed Corn-, Wheat-, or Barley-Based Diets by Chaperonin-60 Sequencing and Quantitative PCR. Appl. Environ. Microbiol. 71: 867-875 [Abstract] [Full Text]  
  • Kurata, S., Kanagawa, T., Magariyama, Y., Takatsu, K., Yamada, K., Yokomaku, T., Kamagata, Y. (2004). Reevaluation and Reduction of a PCR Bias Caused by Reannealing of Templates. Appl. Environ. Microbiol. 70: 7545-7549 [Abstract] [Full Text]  
  • Palumbo, A. V., Schryver, J. C., Fields, M. W., Bagwell, C. E., Zhou, J.-Z., Yan, T., Liu, X., Brandt, C. C. (2004). Coupling of Functional Gene Diversity and Geochemical Data from Environmental Samples. Appl. Environ. Microbiol. 70: 6525-6534 [Abstract] [Full Text]  
  • Lawley, B., Ripley, S., Bridge, P., Convey, P. (2004). Molecular Analysis of Geographic Patterns of Eukaryotic Diversity in Antarctic Soils. Appl. Environ. Microbiol. 70: 5963-5972 [Abstract] [Full Text]  
  • Suchodolski, J. S., Ruaux, C. G., Steiner, J. M., Fetz, K., Williams, D. A. (2004). Application of Molecular Fingerprinting for Qualitative Assessment of Small-Intestinal Bacterial Diversity in Dogs. J. Clin. Microbiol. 42: 4702-4708 [Abstract] [Full Text]  
  • Garcia, B., Olivera, E. R., Sandoval, A., Arias-Barrau, E., Arias, S., Naharro, G., Luengo, J. M. (2004). Strategy for Cloning Large Gene Assemblages as Illustrated Using the Phenylacetate and Polyhydroxyalkanoate Gene Clusters. Appl. Environ. Microbiol. 70: 5019-5025 [Abstract] [Full Text]  
  • Boucher, Y., Douady, C. J., Sharma, A. K., Kamekura, M., Doolittle, W. F. (2004). Intragenomic Heterogeneity and Intergenomic Recombination among Haloarchaeal rRNA Genes. J. Bacteriol. 186: 3980-3990 [Abstract] [Full Text]  
  • Young, L., Dong, Q. (2004). Two-step total gene synthesis method. Nucleic Acids Res 32: e59-e59 [Abstract] [Full Text]  
  • Steward, G. F., Jenkins, B. D., Ward, B. B., Zehr, J. P. (2004). Development and Testing of a DNA Macroarray To Assess Nitrogenase (nifH) Gene Diversity. Appl. Environ. Microbiol. 70: 1455-1465 [Abstract] [Full Text]  
  • Zhou, J., Xia, B., Huang, H., Palumbo, A. V., Tiedje, J. M. (2004). Microbial Diversity and Heterogeneity in Sandy Subsurface Soils. Appl. Environ. Microbiol. 70: 1723-1734 [Abstract] [Full Text]  
  • Smith, A. H., Mackie, R. I. (2004). Effect of Condensed Tannins on Bacterial Diversity and Metabolic Activity in the Rat Gastrointestinal Tract. Appl. Environ. Microbiol. 70: 1104-1115 [Abstract] [Full Text]  
  • Sheehan, K. B., Fagg, J. A., Ferris, M. J., Henson, J. M. (2003). PCR Detection and Analysis of the Free-Living Amoeba Naegleria in Hot Springs in Yellowstone and Grand Teton National Parks. Appl. Environ. Microbiol. 69: 5914-5918 [Abstract] [Full Text]  
  • Liu, X., Bagwell, C. E., Wu, L., Devol, A. H., Zhou, J. (2003). Molecular Diversity of Sulfate-Reducing Bacteria from Two Different Continental Margin Habitats. Appl. Environ. Microbiol. 69: 6073-6081 [Abstract] [Full Text]  
  • Taton, A., Grubisic, S., Brambilla, E., De Wit, R., Wilmotte, A. (2003). Cyanobacterial Diversity in Natural and Artificial Microbial Mats of Lake Fryxell (McMurdo Dry Valleys, Antarctica): a Morphological and Molecular Approach. Appl. Environ. Microbiol. 69: 5157-5169 [Abstract] [Full Text]  
  • Liu, X., Tiquia, S. M., Holguin, G., Wu, L., Nold, S. C., Devol, A. H., Luo, K., Palumbo, A. V., Tiedje, J. M., Zhou, J. (2003). Molecular Diversity of Denitrifying Genes in Continental Margin Sediments within the Oxygen-Deficient Zone off the Pacific Coast of Mexico. Appl. Environ. Microbiol. 69: 3549-3560 [Abstract] [Full Text]  
  • Ferris, M. J., Kuhl, M., Wieland, A., Ward, D. M. (2003). Cyanobacterial Ecotypes in Different Optical Microenvironments of a 68{degrees}C Hot Spring Mat Community Revealed by 16S-23S rRNA Internal Transcribed Spacer Region Variation. Appl. Environ. Microbiol. 69: 2893-2898 [Abstract] [Full Text]  
  • Jenne, C. N., Kennedy, L. J., McCullagh, P., Reynolds, J. D. (2003). A New Model of Sheep Ig Diversification: Shifting the Emphasis Toward Combinatorial Mechanisms and Away from Hypermutation. J. Immunol. 170: 3739-3750 [Abstract] [Full Text]  
  • Schnabel, A., McDonel, P. E., Wendel, J. F. (2003). Phylogenetic relationships in Gleditsia (Leguminosae) based on ITS sequences. Am. J. Bot. 90: 310-320 [Abstract] [Full Text]  
  • Hugenholtz, P., Huber, T. (2003). Chimeric 16S rDNA sequences of diverse origin are accumulating in the public databases. Int. J. Syst. Evol. Microbiol. 53: 289-293 [Abstract] [Full Text]  
  • Nubel, U., Bateson, M. M., Vandieken, V., Wieland, A., Kuhl, M., Ward, D. M. (2002). Microscopic Examination of Distribution and Phenotypic Properties of Phylogenetically Diverse Chloroflexaceae-Related Bacteria in Hot Spring Microbial Mats. Appl. Environ. Microbiol. 68: 4593-4603 [Abstract] [Full Text]  
  • Stevenson, B. (2002). Borrelia burgdorferi erp (ospE-Related) Gene Sequences Remain Stable during Mammalian Infection. Infect. Immun. 70: 5307-5311 [Abstract] [Full Text]  
  • Dunbar, J., Barns, S. M., Ticknor, L. O., Kuske, C. R. (2002). Empirical and Theoretical Bacterial Diversity in Four Arizona Soils. Appl. Environ. Microbiol. 68: 3035-3045 [Abstract] [Full Text]  
  • Hill, J. E., Seipp, R. P., Betts, M., Hawkins, L., Van Kessel, A. G., Crosby, W. L., Hemmingsen, S. M. (2002). Extensive Profiling of a Complex Microbial Community by High-Throughput Sequencing. Appl. Environ. Microbiol. 68: 3055-3066 [Abstract] [Full Text]  
  • Thompson, J. R., Marcelino, L. A., Polz, M. F. (2002). Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by 'reconditioning PCR'. Nucleic Acids Res 30: 2083-2088 [Abstract] [Full Text]  
  • Zhu, X. Y., Zhong, T., Pandya, Y., Joerger, R. D. (2002). 16S rRNA-Based Analysis of Microbiota from the Cecum of Broiler Chickens. Appl. Environ. Microbiol. 68: 124-137 [Abstract] [Full Text]  
  • Zhou, J., Xia, B., Treves, D. S., Wu, L.-Y., Marsh, T. L., O'Neill, R. V., Palumbo, A. V., Tiedje, J. M. (2002). Spatial and Resource Factors Influencing High Microbial Diversity in Soil. Appl. Environ. Microbiol. 68: 326-334 [Abstract] [Full Text]  
  • Wu, L., Thompson, D. K., Li, G., Hurt, R. A., Tiedje, J. M., Zhou, J. (2001). Development and Evaluation of Functional Gene Arrays for Detection of Selected Genes in the Environment. Appl. Environ. Microbiol. 67: 5780-5790 [Abstract] [Full Text]  
  • McSpadden Gardener, B. B., Weller, D. M. (2001). Changes in Populations of Rhizosphere Bacteria Associated with Take-All Disease of Wheat. Appl. Environ. Microbiol. 67: 4414-4425 [Abstract] [Full Text]  
  • Cook, M. A., Osborn, A. M., Bettandorff, J., Sobecky, P. A. (2001). Endogenous isolation of replicon probes for assessing plasmid ecology of marine sediment microbial communities. Microbiology 147: 2089-2101 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»