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Highly thermostable RadA protein from the archaeon Pyrococcus woesei enhances specificity of simplex and multiplex PCR assays

  • Microbial Genetics • Original Paper
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Abstract

The radA gene of the hyperthermophilic archaeon Pyrococcus woesei (Thermococcales) was cloned and overexpressed in Escherichia coli. The 1050-bp gene codes for a 349-amino-acid polypeptide with an M r of 38,397 which shows 100 % positional amino acid identity to Pyrococcus furiosus RadA and 27.1 % to the E. coli RecA protein. Recombinant RadA was overproduced in Escherichia coli as a His-tagged fusion protein and purified to electrophoretic homogeneity using a simple procedure consisting of ammonium sulfate precipitation and metal-affinity chromatography. In solution RadA exists as an undecamer (11-mer). The protein binds both to ssDNA and dsDNA. RadA has been found to be highly thermostable, it remains almost unaffected by a 4-h incubation at 94 °C. The addition of the RadA protein to either simplex or multiplex PCR assays, significantly improves the specificity of DNA amplification by eliminating non-specific products. Among applications tested the RadA protein proved to be useful in allelic discrimination assay of HADHA gene associated with long-chain 3-hydroxylacyl-CoA dehydrogenase deficiency that in infancy may lead to hypotonia, serious heart and liver problems and even sudden death.

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Acknowledgments

The authors would like to thank the staff of Department of Microbiology and A&A Biotechnology for their support for this study.

This work was supported by funding from the European Union’s Seventh Framework Programme managed by REA, Research Executive Agency http://ec.europa.eu/research/rea ([FP7/2007-2013] [FP7/2007-2011]) under grant agreement no 286556 to the EXGENOME project (Exgenome Molecular Enzymes).

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Microbiology, University of Gdansk, Wita Stwosza 59, 80-308, Gdansk, Poland

    Aleksandra Stefanska, Lidia Gaffke, Magdalena Plotka & Tadeusz Kaczorowski

  2. Collection of Plasmids and Microorganisms, University of Gdansk, Wita Stwosza 59, 80-308, Gdansk, Poland

    Anna-Karina Kaczorowska

  3. A&A Biotechnology, Aleja Zwyciestwa 96/98, 81-451, Gdynia, Poland

    Slawomir Dabrowski

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  1. Aleksandra Stefanska
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  2. Lidia Gaffke
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  3. Anna-Karina Kaczorowska
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  4. Magdalena Plotka
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  5. Slawomir Dabrowski
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  6. Tadeusz Kaczorowski
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Correspondence to Tadeusz Kaczorowski.

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Communicated by: Agnieszka Szalewska-Palasz

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Stefanska, A., Gaffke, L., Kaczorowska, AK. et al. Highly thermostable RadA protein from the archaeon Pyrococcus woesei enhances specificity of simplex and multiplex PCR assays. J Appl Genetics 57, 239–249 (2016). https://doi.org/10.1007/s13353-015-0314-5

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  • DOI: https://doi.org/10.1007/s13353-015-0314-5

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