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.
Similar content being viewed by others
References
Valasek MA, Repa JJ (2005) Adv Physiol Educ 29:151
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
OECD BioTrack Database (2008) http://www2.oecd.org/biotech. Accessed 20 Nov 2008
Clive J (2006) ISAAA, International Service for the Acquisition of Agri-Biotech Applications, Brief No 35. http://www.isaaa.org. Accessed 20 Nov 2008
Walsh PS, Erlich HA, Higuchi R (1992) Genome Res 1:241
Schäferling M, Nagl S (2006) Anal Bioanal Chem 385:500
Weiler J, Gausepohl H, Hauser N et al (1997) Nucleic Acids Res 25:2792
Guo Z, Guilfoyle RA, Thiel AJ et al (1994) Nucleic Acids Res 22:5456
Egholm M, Buchardt O, Christensen L et al (1993) Nature 365:566
Ray A, Norden B (2000) FASEB J 14:1041
Lucarelli F, Tombelli S, Minunni M et al (2008) Anal Chim Acta 609:139
Schweitzer B, Kingsmore S (2001) Curr Opin Biotechnol 12:21
Fritzsche W, Taton TA (2003) Nanotechnology 14:R63
Patolsky F, Lichtenstein A, Willner I (2001) Nat Biotechnol 19:253
Bao YP, Huber M, Wei TF et al (2005) Nucleic Acids Res 33:E15
Minunni M, Tombelli S, Fonti J et al (2005) J Am Chem Soc 127:7966
Sims PW, Vasser M, Wong WL et al (2000) Anal Biochem 281:230
Barletta J (2006) Mol Aspects Med 27:224
Niemeyer CM, Adler M, Wacker R (2005) Trends Biotechnol 23:208
Niemeyer CM, Adler M, Pignataro B et al (1999) Nucleic Acids Res 27:4553
Holck A, Va M, Didierjean L et al (2002) Eur Food Res Technol 214:449
Yao D, Yu F, Kim J et al (2004) Nucleic Acids Res 32:E177
Liu J, Tian S, Nielsen PE et al (2005) Chem Commun 23:2969
Corradini R, Feriotto G, Sforza S et al (2004) J Mol Recognit 17:76
Wang R, Minunni M, Tombelli S et al (2004) Biosens Bioelectron 20:598
Adler M, Wacker R, Niemeyer CM (2008) Analyst 133:702
Brandt O, Feldner J, Hellweg S et al (2006) Appl Surf Sci 252:6935
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.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
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
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-009-2724-x