Increased single-nucleotide discrimination of PCR by primer probes bearing hydrophobic 4'C modifications

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2004
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Strerath, Michael
Gaster, Jens
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ChemBioChem. 2004, 5(3), pp. 333-339. ISSN 1439-4227. Available under: doi: 10.1002/cbic.200300757
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We report on significantly increased selectivity of real-time PCR through employment of primer probes that bear hydrophobic 4'C modifications at the 3'-terminal nucleotide. The primer probes were designed to bind the target sequences in such a way that the 3'-terminal nucleotide defines whether a matched or a single mismatched basepair is present depending on the respective target sequence. Several commercially available thermostable DNA polymerases belonging to different DNA polymerase families were tested for their efficacy in discriminating between PCR amplification of matched substrates and duplexes that contain a single mismatch. It turned out that, depending on the 4'C modification and the employed DNA polymerase, significantly increased differentiation between single matches and mismatches could be observed with real-time PCR. The degrees of the observed effects varied with the employed 4'C modification and the sequence context studied. The system is robust enough to work faithfully under several buffer conditions. Our approach should be useful for the direct diagnosis of single nucleotide variations within genes, like single nucleotide polymorphisms or mutations, by PCR without the need for further time- and cost-intensive post-PCR analysis.

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ISO 690STRERATH, Michael, Jens GASTER, Daniel SUMMERER, Andreas MARX, 2004. Increased single-nucleotide discrimination of PCR by primer probes bearing hydrophobic 4'C modifications. In: ChemBioChem. 2004, 5(3), pp. 333-339. ISSN 1439-4227. Available under: doi: 10.1002/cbic.200300757
BibTex
@article{Strerath2004-03-05Incre-351,
  year={2004},
  doi={10.1002/cbic.200300757},
  title={Increased single-nucleotide discrimination of PCR by primer probes bearing hydrophobic 4'C modifications},
  number={3},
  volume={5},
  issn={1439-4227},
  journal={ChemBioChem},
  pages={333--339},
  author={Strerath, Michael and Gaster, Jens and Summerer, Daniel and Marx, Andreas}
}
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