Analytical and Bioanalytical Chemistry

, Volume 406, Issue 15, pp 3755–3762 | Cite as

Specific enrichment of prokaryotic DNA using a recombinant DNA-binding protein

  • Natalia SandetskayaEmail author
  • Andreas Naumann
  • Katharina Hennig
  • Dirk Kuhlmeier
Research Paper


Targeted enrichment of DNA is often necessary for its detection and characterization in complex samples. We describe the development and application of the novel molecular tool for the specific enrichment of prokaryotic DNA. A fused protein comprising the DNA-binding subunit of the bacterial topoisomerase II, gyrase, was expressed, purified, and immobilized on magnetic particles. We demonstrated the specific affinity of the immobilized protein towards bacterial DNA and investigated its efficiency in the samples with high background of eukaryotic DNA. The reported approach allowed for the selective isolation and further detection of as few as 5 pg Staphylococcus aureus DNA from the sample with 4 × 106-fold surplus of human DNA. This method is a promising approach for the preparation of such type of samples, for example in molecular diagnostics of sepsis.


Gyrase Bacterial DNA Target enrichment Bacteria detection 


  1. 1.
    Ghosh I, Stains CI, Ooi AT, Segal DJ (2006) Direct detection of double-stranded DNA: molecular methods and applications for DNA diagnostics. Mol Biosyst 2:551–560CrossRefGoogle Scholar
  2. 2.
    Helwa R, Hoheisel JD (2010) Analysis of DNA-protein interactions: from nitrocellulose filter binding assays to microarray studies. Anal Bioanal Chem 398:2551–2561CrossRefGoogle Scholar
  3. 3.
    Zheng Z, Wang Y (2011) DNA binding proteins: outline of functional classification. BioMolecular Concepts 2:293–303CrossRefGoogle Scholar
  4. 4.
    Lee DK, Seol W, Kim JS (2003) Custom DNA-binding proteins and artificial transcription factors. Curr Top Med Chem 3:645–657CrossRefGoogle Scholar
  5. 5.
    Dhanasekaran M, Negi S, Sugiura Y (2006) Designer zinc finger proteins: tools for creating artificial DNA-binding functional proteins. Acc Chem Res 39:45–52CrossRefGoogle Scholar
  6. 6.
    Nagaoka M, Sugiura Y (2000) Artificial zinc finger peptides: creation, DNA recognition, and gene regulation. J Inorg Biochem 82:57–63CrossRefGoogle Scholar
  7. 7.
    Disqué C (2007) Einfluss der DNA-Extraktion auf die PCR-Detektion von Sepsiserregern (publication in German). BIOspektrum 6:627–629Google Scholar
  8. 8.
    Handschur M, Karlic H, Hertel C, Pfeilstöcker M, Haslberger AG (2009) Preanalytic removal of human DNA eliminates false signals in general 16S rDNA PCR monitoring of bacterial pathogens in blood. Comp Immunol Microbiol Infect Dis 32:207–219CrossRefGoogle Scholar
  9. 9.
    Hansen WL, Bruggeman CA, Wolffs PF (2009) Evaluation of new preanalysis sample treatment tools and DNA isolation protocols to improve bacterial pathogen detection in whole blood. J Clin Microbiol 47:2629–2631CrossRefGoogle Scholar
  10. 10.
    Loonen AJM, Bos MP, van Meerbergen B, Neerken S, Catsburg A, Dobbelaer I, Penterman R, Maertens G, van de Wiel P, Savelkoul P, van den Brule AJC (2013) Comparison of pathogen DNA isolation methods from large volumes of whole blood to improve molecular diagnosis of bloodstream infections. PLoS ONE 8:e72349. doi: 10.1371/journal.pone.0072349 CrossRefGoogle Scholar
  11. 11.
    Yang Y, Ames GF (1988) DNA gyrase binds to the family of prokaryotic repetitive extragenic palindromic sequences. Proc Natl Acad Sci U S A 85:8850–8854CrossRefGoogle Scholar
  12. 12.
    Baker NM, Weigand S, Maar-Mathias S, Mondragon A (2011) Solution structures of DNA-bound gyrase. Nucleic Acids Res 39:755–766CrossRefGoogle Scholar
  13. 13.
    Higgins NP, Cozzarelli NR (1982) The binding of gyrase to DNA: analysis by retention by nitrocellulose filters. Nucleic Acids Res 10:6833–6847CrossRefGoogle Scholar
  14. 14.
    Nöllmann M, Crisona NJ, Arimondo PB (2007) Thirty years of Escherichia coli DNA gyrase: from in vivo function to single-molecule mechanism. Biochimie 89:490–499CrossRefGoogle Scholar
  15. 15.
    Reece RJ, Maxwell A (1991) The C-terminal domain of the Escherichia coli DNA gyrase A subunit is a DNA-binding protein. Nucleic Acids Res 19:1399–1405CrossRefGoogle Scholar
  16. 16.
    Reece RJ, Maxwell A (1991) DNA gyrase: structure and function. Crit Rev Biochem Mol Biol 26:335–375CrossRefGoogle Scholar
  17. 17.
    Lanz MA, Klostermeier D (2012) The GyrA-box determines the geometry of DNA bound to gyrase and couples DNA binding to the nucleotide cycle. Nucleic Acids Res 40:10893–10903CrossRefGoogle Scholar
  18. 18.
    Klaussegger A, Hell M, Berger A, Zinober K, Baier S, Jones N, Sperl W, Kofler B (1999) Gram type-specific broad-range PCR amplification for rapid detection of 62 pathogenic bacteria. J Clin Microbiol 37:464–466Google Scholar
  19. 19.
    McCurdy RD, McGrath JJ, Mackay-Sim A (2008) Validation of the comparative quantification method of real-time PCR analysis and a cautionary tale of housekeeping gene selection. Gene Ther Mol Biol 12:15–24Google Scholar
  20. 20.
    Maheux AF, Picard FJ, Boissinot M, Bissonnette L, Paradis S, Bergeron MG (2009) Analytical comparison of nine PCR primer sets designed to detect the presence of Escherichia coli/Shigella in water samples. Water Res 43:3019–3028CrossRefGoogle Scholar
  21. 21.
    Brakstad OG, Aasbakk K, Maeland JA (1992) Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J Clin Microbiol 30:1654–1660Google Scholar
  22. 22.
    Caroll N, Adamson P, Okhravi N (1999) Elimination of bacterial DNA from TaqDNA polymerases by restriction endonuclease digestion. J Clin Microbiol 37:3402–3404Google Scholar
  23. 23.
    Corless CE, Guiver M, Borrow R, Edwards-Jones V, Kaczmarski EB, Fox AJ (2000) Contamination and sensitivity issues with a realtime universal 16S rRNA PCR. J Clin Microbiol 38:1747–1752Google Scholar
  24. 24.
    Meier A, Persing DH, Finken M, Böttger EC (1993) Elimination of contaminating DNA within polymerase chain reaction reagents: implications for a general approach to detection of uncultured pathogens. J Clin Microbiol 31:646–652Google Scholar
  25. 25.
    Mühl H, Kochem AJ, Disqué C, Sakka SG (2010) Activity and DNA contamination of commercial polymerase chain reaction reagents for the universal 16S rDNA real-time polymerase chain reaction detection of bacterial pathogens in blood. Diagn Microbiol Infect Dis 66:41–49CrossRefGoogle Scholar
  26. 26.
    Klaschik S, Lehmann LE, Raadts A, Hoeft A, Stuber F (2002) Comparison of different decontamination methods for reagents to detect low concentrations of bacterial 16S DNA by real-time-PCR. Mol Biotechnol 22:231–242CrossRefGoogle Scholar
  27. 27.
    Niimi H, Mori M, Tabata H, Minami H, Ueno T, Hayashi S, Kitajima I (2011) A novel eukaryote-made thermostable DNA polymerase which is free from bacterial DNA contamination. J Clin Microbiol 49:3316–3320CrossRefGoogle Scholar
  28. 28.
    Van Meerbergen B, Piciu OM, Gill R, Schmidt KA, Neerken S, Ponjee M, Unay ZS, Penterman R, van de Wiel P (2011) Selective lysis of cells. WIPO Patent Application WO/2011/070507Google Scholar
  29. 29.
    Cox RA (2004) Quantitative relationships for specific growth rates and macromolecular compositions of Mycobacterium tuberculosis, Streptomyces coelicolor A3(2) and Escherichia coli B/r: an integrative theoretical approach. Microbiology 150:1413–1426CrossRefGoogle Scholar
  30. 30.
    Vieira MS (1999) Statistics of DNA sequences: a low-frequency analysis. Phys Rev 60:5932–5937Google Scholar
  31. 31.
    Mangiapan G, Vokurka M, Schouls L, Cadranel J, Lecossier D, van Embden J, Hance AJ (1996) Sequence capture-PCR improves detection of mycobacterial DNA in clinical specimens. J Clin Microbiol 34:1209–1215Google Scholar
  32. 32.
    Parham NJ, Picard FJ, Peytavi R, Gagnon M, Seyrig G, Gagné PA, Boissinot M, Bergeron MG (2007) Specific magnetic bead based capture of genomic DNA from clinical samples: application to the detection of group B streptococci in vaginal/anal swabs. Clin Chem 53:1570–1576CrossRefGoogle Scholar
  33. 33.
    Peeters S, Stakenborg T, Colle F, Liu C, Lagae L, Van Ranst M (2010) Real-time PCR to study the sequence specific magnetic purification of DNA. Biotechnol Prog 26:1678–1684CrossRefGoogle Scholar
  34. 34.
    Sachse S, Straube E, Lehmann M, Bauer M, Russwurm S, Schmidt KH (2009) Truncated human cytidylate-phosphate-deoxyguanylate-binding protein for improved nucleic acid amplification technique-based detection of bacterial species in human samples. J Clin Microbiol 47:1050–1057CrossRefGoogle Scholar
  35. 35.
    Turek-Plewa J, Jagodziński PP (2005) The role of mammalian DNA methyltransferases in the regulation of gene expression. Cell Mol Biol Lett 10:631–647Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Natalia Sandetskaya
    • 1
    Email author
  • Andreas Naumann
    • 1
  • Katharina Hennig
    • 2
  • Dirk Kuhlmeier
    • 1
  1. 1.Nanotechnology UnitFraunhofer Institute for Cell Therapy and Immunology IZILeipzigGermany
  2. 2.University of Applied Sciences JenaJenaGermany

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