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A novel approach for the detection of DNA using immobilized peptide nucleic acid (PNA) probes and signal enhancement by real-time immuno-polymerase chain reaction (RT-iPCR)

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Abstract

A new approach for the detection of DNA target molecules is described, using capture probes and subsequent signal enhancement by a uniform polymerase chain reaction (PCR). Peptide nucleic acid probes were immobilized in real-time PCR-compatible microtiter plates. After hybridization of biotinylated DNA targets, detection was performed by real-time immuno-PCR, a method formerly used for protein detection. We demonstrate the feasibility of this strategy for the qualitative detection of DNA oligonucleotides with a detection limit (LOD) of 6 attomol. Furthermore, the method was applied to PCR-amplified samples from genetically modified maize DNA (Mon810). A 483-bp DNA fragment was detected in mixture with 99.9% of noncomplementary DNA with a sensitivity down to the level of attomole.

 

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References

  1. Valasek MA, Repa JJ (2005) Adv Physiol Educ 29:151

    Article  Google Scholar 

  2. EC (2008) Community register of genetically modified food and feed. http://ec.europa.eu/food/dyna/gm_register/index_en.cfm. Accessed 20 Nov 2008

  3. OECD BioTrack Database (2008) http://www2.oecd.org/biotech. Accessed 20 Nov 2008

  4. Clive J (2006) ISAAA, International Service for the Acquisition of Agri-Biotech Applications, Brief No 35. http://www.isaaa.org. Accessed 20 Nov 2008

  5. Walsh PS, Erlich HA, Higuchi R (1992) Genome Res 1:241

    Article  CAS  Google Scholar 

  6. Schäferling M, Nagl S (2006) Anal Bioanal Chem 385:500

    Article  Google Scholar 

  7. Weiler J, Gausepohl H, Hauser N et al (1997) Nucleic Acids Res 25:2792

    Article  CAS  Google Scholar 

  8. Guo Z, Guilfoyle RA, Thiel AJ et al (1994) Nucleic Acids Res 22:5456

    Article  CAS  Google Scholar 

  9. Egholm M, Buchardt O, Christensen L et al (1993) Nature 365:566

    Article  CAS  Google Scholar 

  10. Ray A, Norden B (2000) FASEB J 14:1041

    CAS  Google Scholar 

  11. Lucarelli F, Tombelli S, Minunni M et al (2008) Anal Chim Acta 609:139

    Article  CAS  Google Scholar 

  12. Schweitzer B, Kingsmore S (2001) Curr Opin Biotechnol 12:21

    Article  CAS  Google Scholar 

  13. Fritzsche W, Taton TA (2003) Nanotechnology 14:R63

    Article  CAS  Google Scholar 

  14. Patolsky F, Lichtenstein A, Willner I (2001) Nat Biotechnol 19:253

    Article  CAS  Google Scholar 

  15. Bao YP, Huber M, Wei TF et al (2005) Nucleic Acids Res 33:E15

    Article  Google Scholar 

  16. Minunni M, Tombelli S, Fonti J et al (2005) J Am Chem Soc 127:7966

    Article  CAS  Google Scholar 

  17. Sims PW, Vasser M, Wong WL et al (2000) Anal Biochem 281:230

    Article  CAS  Google Scholar 

  18. Barletta J (2006) Mol Aspects Med 27:224

    Article  CAS  Google Scholar 

  19. Niemeyer CM, Adler M, Wacker R (2005) Trends Biotechnol 23:208

    Article  CAS  Google Scholar 

  20. Niemeyer CM, Adler M, Pignataro B et al (1999) Nucleic Acids Res 27:4553

    Article  CAS  Google Scholar 

  21. Holck A, Va M, Didierjean L et al (2002) Eur Food Res Technol 214:449

    Article  CAS  Google Scholar 

  22. Yao D, Yu F, Kim J et al (2004) Nucleic Acids Res 32:E177

    Article  Google Scholar 

  23. Liu J, Tian S, Nielsen PE et al (2005) Chem Commun 23:2969

    Article  Google Scholar 

  24. Corradini R, Feriotto G, Sforza S et al (2004) J Mol Recognit 17:76

    Article  CAS  Google Scholar 

  25. Wang R, Minunni M, Tombelli S et al (2004) Biosens Bioelectron 20:598

    Article  CAS  Google Scholar 

  26. Adler M, Wacker R, Niemeyer CM (2008) Analyst 133:702

    Article  CAS  Google Scholar 

  27. Brandt O, Feldner J, Hellweg S et al (2006) Appl Surf Sci 252:6935

    Article  CAS  Google Scholar 

Download references

Acknowledgment

This work was financially supported by the European Commission through the Integrated Project “Co-Extra”, contract 2003-7158, under the 6th Framework Programme, priority 5, food quality and safety. This support is gratefully acknowledged.

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Authors and Affiliations

  1. Bundesinstitut für Risikobewertung (BfR), Abteilung Lebensmittelsicherheit, Thielallee 88-92, 14195, Berlin, Germany

    Lillian Roth, Jutta Zagon, Anke Ehlers & Hermann Broll

  2. Department of Food Analysis, Institute of Food Technology and Food Chemistry, Technical University of Berlin, Gustav Meyer Allee 25, 13355, Berlin, Germany

    Lothar W. Kroh

Authors
  1. Lillian Roth
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  2. Jutta Zagon
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  3. Anke Ehlers
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  4. Lothar W. Kroh
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  5. Hermann Broll
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Correspondence to Lillian Roth.

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Roth, L., Zagon, J., Ehlers, A. et al. A novel approach for the detection of DNA using immobilized peptide nucleic acid (PNA) probes and signal enhancement by real-time immuno-polymerase chain reaction (RT-iPCR). Anal Bioanal Chem 394, 529–537 (2009). https://doi.org/10.1007/s00216-009-2724-x

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  • DOI: https://doi.org/10.1007/s00216-009-2724-x

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