Evaluation of PCR amplification bias by terminal restriction fragment length polymorphism analysis of small-subunit rRNA and mcrA genes by using defined template mixtures of methanogenic pure cultures and soil DNA extracts

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a widely used method for profiling microbial community structure in different habitats by targeting small-subunit (SSU) rRNA and also functional marker genes. It is not known, however, whether relative gene frequencies of individ...

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Vydané v:Applied and environmental microbiology Ročník 69; číslo 1; s. 320
Hlavní autori: Lueders, Tillmann, Friedrich, Michael W
Médium: Journal Article
Jazyk:English
Vydavateľské údaje: United States 01.01.2003
Predmet:
Bias
Culture Media
DNA, Bacterial - analysis
DNA, Bacterial - isolation & purification
Ecosystem
Euryarchaeota - classification
Euryarchaeota - genetics
Euryarchaeota - growth & development
Oxidoreductases - genetics
Polymerase Chain Reaction - methods
Polymorphism, Restriction Fragment Length
Reproducibility of Results
RNA, Ribosomal - genetics
Soil - analysis
Templates, Genetic
ISSN:0099-2240
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Shrnutí:Terminal restriction fragment length polymorphism (T-RFLP) analysis is a widely used method for profiling microbial community structure in different habitats by targeting small-subunit (SSU) rRNA and also functional marker genes. It is not known, however, whether relative gene frequencies of individual community members are adequately represented in post-PCR amplicon frequencies as shown by T-RFLP. In this study, precisely defined artificial template mixtures containing genomic DNA of four different methanogens in various ratios were prepared for subsequent T-RFLP analysis. PCR amplicons were generated from defined mixtures targeting not only the SSU rRNA but also the methyl-coenzyme M reductase (mcrA/mrtA) genes of methanogens. Relative amplicon frequencies of microorganisms were quantified by comparing fluorescence intensities of characteristic terminal restriction fragments. SSU ribosomal DNA (rDNA) template ratios in defined template mixtures of the four-membered community were recovered absolutely by PCR-T-RFLP analysis, which demonstrates that the T-RFLP analysis evaluated can give a quantitative view of the template pool. SSU rDNA-targeted T-RFLP analysis of a natural community was found to be highly reproducible, independent of PCR annealing temperature, and unaffected by increasing PCR cycle numbers. Ratios of mcrA-targeted T-RFLP analysis were biased, most likely by PCR selection due to the degeneracy of the primers used. Consequently, for microbial community analyses, each primer system used should be evaluated carefully for possible PCR bias. In fact, such bias can be detected by using T-RFLP analysis as a tool for the precise quantification of the PCR product pool.
Bibliografia:ObjectType-Article-2
SourceType-Scholarly Journals-1
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ISSN:0099-2240
DOI:10.1128/AEM.69.1.320-326.2003