Evaluation of single-nucleotide primer extension for detection and typing of phylogenetic markers used for investigation of microbial communities.
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.
Nikolausz
Marcell
M
UFZ Helmholtz Centre for Environmental Research, Department of Environmental Biotechnology, Permoserstr. 15, 04318 Leipzig, Germany. marcell.nikolausz@ufz.de
Chatzinotas
Antonis
A
Táncsics
András
A
Imfeld
Gwenaël
G
Kästner
Matthias
M
D001419:Bacteria / Q000145:classification* / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics*; D004275:DNA, Ribosomal / Q000235:genetics*; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S