A new assay for single nucleotide polymorphism analysis based on displacement reactions in PNA–DNA double helices

Abstract

In this article, we describe a new method for single nucleotide polymorphism analysis using the displacement reaction of the DNA in a PNA–DNA double helix by the target DNA. Thereby, the probe consists of a TMR-labeled PNA and a Cy5-labeled DNA forming a FRET system. Due to the displacement of the labeled DNA strand by the target DNA, the FRET is invalidated and the fluorescence of the donor dye (TMR) increases. Investigations of the exchange reaction show that increasing salt concentration and temperature accelerate the exchange rate.

Keywords:

• PNA–DNA systems
• Fluorescence-resonance-energy-transfer
• Single nucleotide polymorphism analysis
• DNA assay

Acknowledgements

We thank Prof. Roland Krämer (Institute of Inorganic Chemistry, University of Heidelberg) for the PNA-synthesis and Prof. Jürgen Wolfrum and Dr Jörg Hoheisel for their support and discussions. For financial support the Landesstiftung Baden-Württemberg is acknowledged.