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 - PubMed (original) (raw)

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

Tillmann Lueders et al. Appl Environ Microbiol. 2003 Jan.

Abstract

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.

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Figures

FIG. 1.

FIG. 1.

Effect of various PCR annealing temperatures on the SSU rDNA-targeted (A) and _mcrA-_targeted (B) T-RFLP analysis of a soil archaeal community. Amplicons were generated from identical soil DNA templates with various PCR annealing temperatures and analyzed by T-RFLP fingerprinting. RAF, relative amplicon frequency.

FIG. 2.

FIG. 2.

Electropherograms of T-RFLP analysis of defined template mixtures of M. bryantii (MB), M. concilii (MX), M. hungatei (MP), and M. jannaschii (MC) pure-culture DNA. Shown is a series with increasing M. bryantii template ratios from 25 to 78.6% of SSU rRNA genes (A to D). T-RF lengths of characteristic peaks are indicated in parentheses (in base pairs). RFU, relative fluorescence units.

FIG. 3.

FIG. 3.

Relationship between SSU rDNA template ratios and relative amplicon frequencies as determined by T-RFLP analysis of defined template mixtures. Relative amplicon frequencies (RAF) were determined by fluorescence peak height analysis (A) or peak area integration (B). Lines indicate best linear fit for combined data sets of each graph, and slopes are given. Average values of separately prepared defined template mixtures are plotted (± standard deviation, n = 3).

FIG. 4.

FIG. 4.

Relationship between SSU rDNA and mcrA/mrtA gene relative amplicon frequencies (RAF) as determined by T-RFLP analysis of defined template mixtures. Lines indicate estimated fits.

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