Fӧrster resonance energy transfer sensor with a quantum dot
for detection of specific DNA sequences
Vladimíra Datinská1,2, František Foret1, Karel Klepárník1
1
Institute of Analytical Chemistry of the CAS v.v.i, (Veveří 97, Brno, CZ-602 00)
2
Masaryk University, Faculty of Science (Kotlářská 267/2, Brno, CZ-611 37)
datinska@iach.cz
Abstract:
The synthesis, structure and application of a Fӧrster resonance energy transfer (FRET)
sensor for the detection of specific DNA sequences are investigated. The sensor is
intended for detection of circulation tumor DNA from blood samples. Therefore, we
also focused on sample preparation of nucleic acids.
The sensor is based on the hybridization of oligonucleotide modified with quantum
dots (QDs) and a fluorescently labeled sample DNA resulting in changes of the laserinduced
fluorescence emission due to the FRET effect. The analyzed sample DNA is
a nucleotide primer fluorescently labeled at its 3’ end by carboxy-X-rhodamine
(ROX). Upon hybridization of the ssDNA sample with the QD conjugate, the QD and
ROX label get close to each other. After irradiation, part of the energy absorbed by
QD donor is nonradiatively transferred to the ROX acceptor and a decrease in QD
luminescence emission and an increase in ROX fluorescence emission is observed.
The structure of the sensor was determined by the dependence of FRET efficiency on
the concentration of complementary ROX-ssDNA and confirmed by capillary
electrophoresis analysis.
Real DNA samples often include a complex matrix (such as blood and other bodily
fluids, or exogenous impurities, e.g., from the scene of crime). Most of the common
nucleic acids isolation techniques are based on extractions; however, isotachophoretic
focusing has recently attracted some interest for its simplicity and potential for very
high enrichment factors and ease of automation. We report on the use of a commercial
isotachophoretic instrument for optimization of DNA and preparative fraction
collection. The sample of a DNA ladder was injected in 30 μl volume and after ITP
focusing the DNA zone was recovered using an on-column micropreparative
collection valve. The DNA content in the collected sample was verified by
fluorescence spectrometry and chip capillary electrophoresis with fluorescence
detection.
Acknowledgement:
This work was supported by the Grant Agency of the Czech Republic project (project
No. 14-28254S and P20612G014) and by Technology Agency of the Czech Republic
(TA02010672). Additional support was by Roche Sequencing Solutions (Pleasanton,
USA) and by Institutional support RVO 68081715 of Institute of Analytical Chemistry,
Czech Academy of Sciences, v.v.i. (Brno, Czech Republic). This research was partly
carried out under the project CEITEC 2020 (LQ1601) with financial support from the
Ministry of Education, Youth and Sports of the Czech Republic under the National
Sustainability Programme II.