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Applied and Environmental Microbiology, May 2009, p. 2850-2860, Vol. 75, No. 9
0099-2240/09/$08.00+0     doi:10.1128/AEM.01910-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Evaluation of Single-Nucleotide Primer Extension for Detection and Typing of Phylogenetic Markers Used for Investigation of Microbial Communities{triangledown}

Marcell Nikolausz,1* Antonis Chatzinotas,2 András Táncsics,3 Gwenaël Imfeld,4 and Matthias Kästner1

UFZ, Helmholtz Centre for Environmental Research, Department of Environmental Biotechnology, Permoserstr. 15, D-04318 Leipzig, Germany,1 UFZ, Helmholtz Centre for Environmental Research, Department of Environmental Microbiology, Permoserstr. 15, D-04318 Leipzig, Germany,2 Eötvös Loránd University of Science, Department of Microbiology, Pázmány Péter Sétány 1/c, 1117 Budapest, Hungary,3 UFZ, Helmholtz Centre for Environmental Research, Department of Isotope Biogeochemistry, Permoserstr. 15, D-04318 Leipzig, Germany4

Received 18 August 2008/ Accepted 20 February 2009

Single-nucleotide primer extension (SNuPE) is an emerging tool for parallel detection of DNA sequences of different target microorganisms. The specificity and sensitivity of the SNuPE method were assessed by performing single and multiplex reactions using defined template mixtures of 16S rRNA gene PCR products obtained from pure bacterial cultures. The mismatch discrimination potential of primer extension was investigated by introducing different single and multiple primer-target mismatches. The type and position of the mismatch had significant effects on the specificity of the assay. While a 3'-terminal mismatch has a considerable effect on the fidelity of the extension reaction, the internal mismatches influenced hybridization mostly by destabilizing the hybrid duplex. Thus, carefully choosing primer-mismatch positions should result in a high signal-to-noise ratio and prevent any nonspecific extension. Cyclic fluorescent labeling of the hybridized primers via extension also resulted in a significant increase in the detection sensitivity of the PCR. In multiplex reactions, the signal ratios detected after specific primer extension correlated with the original template ratios. In addition, reverse-transcribed 16S rRNA was successfully used as a nonamplified template to prove the applicability of SNuPE in a PCR-independent manner. In conclusion, this study demonstrates the great potential of SNuPE for simultaneous detection and typing of various nucleic acid sequences from both environmental and engineered samples.

* Corresponding author. Mailing address: UFZ Helmholtz Centre for Environmental Research, Department of Environmental Biotechnology, Permoserstr. 15, 04318 Leipzig, Germany. Phone: 49 341 235 1763. Fax: 49 341 235 2492. E-mail: marcell.nikolausz{at}ufz.de

{triangledown} Published ahead of print on 27 February 2009.

Applied and Environmental Microbiology, May 2009, p. 2850-2860, Vol. 75, No. 9
0099-2240/09/$08.00+0     doi:10.1128/AEM.01910-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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