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).
Highlights
Sequence-specific hybridization between single-stranded oligonucleotides immobilized on a sensor surface and fragments of nucleic acids of the investigated samples is a straightforward way for identification of various genetic and infectious diseases, especially on their early stages
Completely complementary oligonucleotide mod-Ph is used as a probe for immobilization on the sensor surface
The experimental values of Tm determined for heterogeneous system are a few degrees lower than the values obtained using the web server DINAMelt for the solution-phase hybridization (Table 3)
Summary
Sequence-specific hybridization between single-stranded oligonucleotides immobilized on a sensor surface and fragments of nucleic acids of the investigated samples is a straightforward way for identification of various genetic and infectious diseases, especially on their early stages. Matsishin et al Nanoscale Research Letters (2016) 11:19 discrimination of completely complementary and partially complementary oligonucleotides corresponding to hybrid and normal (non-translocated) nucleotide sequences, respectively. Discrimination of completely complementary and partially complementary DNA duplexes on the sensor surface can be achieved by employing various factors influencing hybridization efficiency, including target length and concentration, temperature, composition of hybridization buffer, posthybridization stringency wash, and a combination of the factors [5, 6]. The application of hybridization suppressors like formamide with a high refractive index, which is out (or nearly out) of the detectable range of most SPR equipments, results in a technical challenge for SPR detection of DNA hybridization [5,6,7,8,9]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
