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Biointerphases

, Volume 1, Issue 4, pp 113–122

Received: 21 August 2006Accepted: 20 September 2006

Abstract

Peptide nucleic acid PNA, a polyamide DNA mimic, has inspired the development of a variety of hybridization-based methods for the detection, quantification, purification, and characterization of nucleic acids owing to the stability of the PNA-DNA duplex. In this work, PNA probes complementary to a specific sequence of Roundup Ready soybean were immobilized onto a sensor surface via a self-assembled matrix employing streptavidin-biotin binding. The specific hybridization of PNA and DNA has been monitored by applying the chromophore-labeled DNA target oligonucleotides to the PNA modified Au sensor surface in real time using surface plasmon field-enhanced fluorescence spectroscopy. The authors demonstrate three kinds of experiments called global, titration, and kinetic analyses for the determination of rate constants for the association kon and dissociation processes koff, and the affinity constant KA of the PNA-DNA duplex formation by fitting the data to a simple Langmuir model. Discrimination of a single base mismatched DNA 15mer target on a 15mer PNA probe was documented, with a difference of the affinity constant of two orders of magnitude. Finally, the affinity constant for the hybridization of a long polymerase chain reaction product 169mer obtained by amplification of DNA extracted from genetically modified soybean reference material has been determined by a kinetic-titration analysis. The results show the influence of a Coulomb barrier at high target surface coverage even for the hybridization to PNA at low ionic strength.

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Autor: Hyeyoung Park - Andrea Germini - Stefano Sforza - Roberto Corradini - Rosangela Marchelli - Wolfgang Knoll

Fuente: https://link.springer.com/







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