SPR Detection and Discrimination of the Oligonucleotides Related to the Normal and the Hybrid bcr-abl Genes by Two Stringency Control Strategies

Abstract

In this study, we applied two stringency control strategies for surface plasmon resonance (SPR) detection of DNA hybridization and discrimination of completely and partially complementary 24-mer sequences. These sequences are specific to the human normal bcr and the hybrid bcr-abl genes, protein products of which are responsible for some leukemia. SPR sensors based on resonance phenomena in nanoscale gold films are well suited for label-free, real-time investigations of the macromolecule interactions. Thermodynamic parameters obtained using the web server DINAMelt allowed supposing the possibility for realization (a) stringency control based on the ionic strength of the hybridization buffer and (b) stringency control based on the temperature elevation. The first one resulted in that the discrimination index of completely complementary and partially complementary oligonucleotides depending on the target concentration varied from 1.3 to 1.8 in 2 × SSC and from 2.0 to 2.9 in 0.5 × SSC. For implementation of the second stringency control strategy, SPR spectrometer measuring flow cell with built-in high-precision temperature control and regulation as well as corresponding software was created. It is shown that the duplexes formed by the immobilized probes mod-Ph and completely complementary oligonucleotides P1 remained without significant changes until ~50 °C, while the duplexes formed with partially complementary oligonucleotide Bcrex14 almost entirely disrupted at 40 °C. Thus, the absolutely effective thermodiscrimination of this pair of oligonucleotides was achieved in this temperature range (40-50 °C).

Keywords: Hybridization biosensor; Ionic strength; SPR; Stringency control; Thermodiscrimination.

Fig. 1

The most likely intermolecular duplexes forming at hybridization of the targets P1, Bcrex14, and TC with the immobilized probe mod-Ph

Fig. 2

SPR sensograms for hybridization between the immobilized probe mod-Ph and the targets P1, Bcrex14, and TC in 2 × SSC (a) and in 0.5 × SSC (b)

Fig. 3

Dependence of the ratio of the sensor response at the P1/mod-Ph hybridization to the sensor response at the Bcrex14/mod-Ph hybridization on the concentration of the targets in two buffer solutions used

Fig. 4

Discrimination of P1 (a) and Bcrex14 (b) using controlled temperature change in the measuring cell of the SPR spectrometer “Plasmon 6”

Fig. 5

The dependence of the ratio of sensor response after hybridization of target oligonucleotide P1 or Bcrex14 with immobilized mod-Ph, heating and cooling to the initial sensor response on the heating temperature