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Cited by 414

The DOI matches its Crossref record; 39 of the 39 references it cites were found in a registry or an index.

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

Open access
Qiu, Xiaoyun, Wu, Liyou, Huang, Heshu, McDonel, Patrick E., Palumbo, Anthony V., Tiedje, James M., Zhou, Jizhong·2001·Applied and Environmental Microbiology
DOI 10.1128/aem.67.2.880-887.2001
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Abstract

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 Hha I 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, Ampli Taq . 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 Ampli Taq (2.5%). The frequencies of chimeras and of heteroduplexes for Z- Taq were almost three times higher than those of Ampli Taq . 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 Ampli Taq 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.

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Metadata

Authors
Qiu, X., Wu, L., Huang, H., McDonel, P. E., Palumbo, A. V., Tiedje, J. M., & Zhou, J.
Venue
Applied and Environmental Microbiology
Publisher
American Society for Microbiology
Published
2001-02
Volume
67
Issue
2
Pages
880-887
ISSN
  • 0099-2240 (ISSN-L)
  • 1098-5336
DOI
10.1128/aem.67.2.880-887.2001
OpenAlex
W2122421045
Cited by
414
References
39
FWCI
7.38
Type
journal article
Language
en
Open access
green

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Qiu, X., Wu, L., Huang, H., McDonel, P. E., Palumbo, A. V., Tiedje, J. M., & Zhou, J. (2001). Evaluation of PCR-Generated Chimeras, Mutations, and Heteroduplexes with 16S rRNA Gene-Based Cloning. Applied and Environmental Microbiology. https://doi.org/10.1128/aem.67.2.880-887.2001

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Contains information from the Semantic Scholar Academic Graph Datasets, made available under the ODC Attribution License (ODC-BY 1.0).

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