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

@article{Qiu2001EvaluationOP,
  title={Evaluation of PCR-Generated Chimeras, Mutations, and Heteroduplexes with 16S rRNA Gene-Based Cloning},
  author={Xiaoyun Qiu and Liyou Wu and Heshu Huang and P. McDonel and A. Palumbo and J. Tiedje and Jizhong Zhou},
  journal={Applied and Environmental Microbiology},
  year={2001},
  volume={67},
  pages={880 - 887}
}
  • Xiaoyun Qiu, Liyou Wu, +4 authors Jizhong Zhou
  • Published 2001
  • Medicine, Biology
  • Applied and Environmental Microbiology
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 Proteobacteriaas 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 TaqDNA polymerases examined: 20% for Z-Taq… Expand
Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by 'reconditioning PCR'.
TLDR
A method to eliminate heteroduplexes from mixed- template PCR products by subjecting them to 'reconditioning PCR', a low cycle number re-amplification of a 10-fold diluted mixed-template PCR product is developed. Expand
The use of unilateral PCR to identify prominent heteroduplexes formed during PCR of the mouse microsatellite locus D17Mit23
TLDR
A modified form of asymmetric PCR is used, called “unilateral PCR”, to show that the higher molecular weight bands are heterodu-plexes and to identify their constituent strands. Expand
Optimized PCR conditions minimizing the formation of chimeric DNA molecules from MPRA plasmid libraries
TLDR
PCR parameters that ensure synthesis of specific (non-chimeric) products from highly diverse MPRA plasmid libraries are identified and there is a negligible difference in performance of emulsion and conventional PCR approaches performed with the identified settings. Expand
Reducing the impact of PCR-mediated recombination in molecular evolution and environmental studies using a new-generation high-fidelity DNA polymerase.
TLDR
This work measures chimera formation by analyzing a mixture of eight partial actin sequences isolated from the amoeba Arcella hemisphaerica amplified under a variety of conditions that mimic standard laboratory situations and confirms that processivity-enhanced polymerases enable a substantial decrease in PCR-mediated recombination through reducing starting template concentration, without compromising the robustness of PCR reactions. Expand
PCR-Induced Sequence Artifacts and Bias: Insights from Comparison of Two 16S rRNA Clone Libraries Constructed from the Same Sample
TLDR
Taq DNA polymerase errors were found to be the dominant sequence artifact but could be constrained by clustering the sequences into 99% sequence similarity groups, and no skew in sequence types was detected in the two libraries constructed from PCR products amplified for different numbers of cycles. Expand
Evaluation of primers and PCR conditions for the analysis of 16S rRNA genes from a natural environment.
TLDR
This work investigated biases occurring in the polymerase chain reaction (PCR) amplification of 16S rRNA genes from an environmental sample by comparing the clone libraries prepared from the gut homogenate of the termite Reticulitermes speratus, and found that the Bacteria-universal primer, 63F, introduced a seriously biased amplification caused by primer mismatches. Expand
Evaluation of Single-Nucleotide Primer Extension for Detection and Typing of Phylogenetic Markers Used for Investigation of Microbial Communities
TLDR
This study demonstrates the great potential of SNuPE for simultaneous detection and typing of various nucleic acid sequences from both environmental and engineered samples. Expand
Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis.
TLDR
The biases associated with preferential amplification of multitemplate PCR were investigated using 'universal' bacteria-specific primers, focusing on the effect of primer mismatch, annealing temperature and PCR cycle number. Expand
Band smearing of PCR amplified bacterial 16S rRNA genes: dependence on initial PCR target diversity.
TLDR
It is considered that with amplified heterogeneous DNA, delayed electro-migration is caused not by PCR errors but by dsDNA structures that arise from imperfect strand pairing, and band smearing occurred due to imperfectly paired strands of the amplified DNA. Expand
Preferential ligation during TA-cloning of multitemplate PCR products--a factor causing bias in microbial community structure analysis.
TLDR
Both TOPO TA and pGem-T vector system cloning kits showed significant and reproducible insert size related selectivity, and the two clone libraries showed significantly different compositions, and were also differing from the community structure revealed by length heterogeneity PCR. Expand
...
1
2
3
4
5
...

References

SHOWING 1-10 OF 43 REFERENCES
PAGE analysis of the heteroduplexes formed between PCR-amplified 16S rRNA genes: estimation of sequence similarity and rDNA complexity.
TLDR
The electrophoretic migration of the heteroduplexes observed after PCR was used to detect the presence of 16S rDNAs with different sequences in DNA extracted from both a mixture of two bacterial species and samples containing a natural bacterial community. Expand
Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients
A group-specific primer, F243 (positions 226 to 243, Escherichia coli numbering), was developed by comparison of sequences of genes encoding 16S rRNA (16S rDNA) for the detection of actinomycetes inExpand
The frequency of chimeric molecules as a consequence of PCR co-amplification of 16S rRNA genes from different bacterial species.
TLDR
This work quantifies the frequency of chimeric sequences in PCR amplification of 16S rRNA genes and examines effects of the number of amplification cycles, length of elongation periods and presence of damaged DNA on chimera formation. Expand
Frequency of formation of chimeric molecules as a consequence of PCR coamplification of 16S rRNA genes from mixed bacterial genomes.
TLDR
The level of occurrence of chimeric sequences after 30 cycles of PCR amplification was 32%, and it is shown that PCR-induced chimeras were formed between different rRNA gene copies from the same organism. Expand
Heteroduplex resolution using T7 endonuclease I in microbial community analyses.
TLDR
Heteroduplexes may be resolved before carrying out genomic library construction by including a digestion step with T7 endonuclease I, and the addition of this step may eliminate heterod uplexes from PCR products and ensure that subsequent genomic libraryConstruction is not compromised. Expand
Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis
TLDR
The results demonstrate the usefulness of TGGE for the structural analysis of heterogeneous rRNA genes together with their expression, stress problems of the generation of meaningful data for 16S rRNA sequences and probe designs, and might have consequences for evolutionary concepts. Expand
16S ribosomal DNA amplification for phylogenetic study
TLDR
By use of this method, the phylogenetic study of extremely fastidious or highly pathogenic bacterial species can be carried out without the need to culture them. Expand
Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA.
A quantitative molecular technique was developed for rapid analysis of microbial community diversity in various environments. The technique employed PCR in which one of the two primers used wasExpand
Nonradioactive method to study genetic profiles of natural bacterial communities by PCR-single-strand-conformation polymorphism
TLDR
A new method based on single-strand-conformation polymorphism (SSCP) analysis of PCR products of 16S rRNA genes from complex bacterial populations provides a useful tool to study bacterial community structures in various ecosystems. Expand
Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR.
TLDR
Three rRNA gene standards were prepared by PCR, mixed in known proportions, and amplified a second time by using primer pairs in which one primer was labeled with a fluorescent nucleotide derivative to fit a kinetic model in which the reannealing of genes progressively inhibits the formation of template-primer hybrids. Expand
...
1
2
3
4
5
...