Detection of a Specific DNA Fragment by Free-Solution Capillary Electrophoresis Using a Fluorescent DNA Probe Measured by Semiconductor Laser-Induced Fluorometry

Abstract

Semiconductor laser-induced fluorescence detection of native (target) DNA has been accomplished by free-solution capillary electrophoresis (CE) using an oligonucleotide labeled with a cyanine dye as a DNA probe. A mixture of probe DNA and target DNA, which is complementary to probe DNA, is incubated to form hybrid DNA; the mixture is then injected into a capillary. Single-stranded probe DNA and double-stranded hybrid DNA were separated by free-solution CE. A 19mer oligonucleotide labeled with fluorescein isothiocyanate (FITC) was used as probe DNA in a preliminary study. The stability of double-stranded DNA during migration was evaluated by changing the temperature of the solution in the capillary, i.e. the applied voltage in CE. The dissociation of double-stranded DNA was appreciable at high applied voltages. Thus, suppressing the temperature in the capillary, i.e. optimization of the voltage, was required in order to prevent the dissociation of double-stranded DNA. Furthermore, a new labeling reagent, a cyanine derivative, was synthesized in order to be applied to semiconductor laser-induced fluorometry. The detection limit was 8X 10-9 M for probe DNA.

Laser‐Induced Fluorometry for Capillary Electrophoresis

“…A diode laser emitting at 780 nm was employed to excite a cyanine‐labeled DNA molecule with the complementary sequence of a target DNA. The cyanine‐labeled DNA acted as the probe DNA for the target DNA by annealing to form the double‐stranded DNA that had been separated from the single‐stranded probe DNA by CE . The limits of detection for the probe DNA was estimated to be 8 nM.…”

Band broadening of DNA fragments isolated by polyacrylamide gel electrophoresis in capillary electrophoresis

“…However, DNA may also be denatured by electrophoresis because double‐stranded DNA generally dissociates at around 60°C, which is the temperature reached by Joule heating generated during electrophoresis. For example, we reported that double‐stranded DNA was dissociated by increasing the separation voltage that leads to a rise in temperature due to Joule heating in capillary electrophoresis (CE) 7. Gao and Yeung also demonstrated that mutations of DNA with a single‐nucleotide polymorphism could be detected by precise temperature control, which permitted manipulation of conformational changes in double‐stranded DNA 8.…”

Separation and Detection of Cyanine-Labeled Amino Acids by Micellar Electrokinetic Chromatography Combined with Fluorescence Detection Using Diode-Based Solid-State Lasers

“…We have reported on analytical applications using diode lasers as excitation sources for CE and micellar electrokinetic chromatography (MEKC) LIF detection of amino acids 9,10 , polycyclic aromatic hydrocarbons 11,12 , and oligonucleotides. 13 One significant limitation of diode lasers as LIF excitation sources is that the laser emission wavelength is too long, and it is difficult to obtain chemical reagents which have absorption bands at such long wavelengths. We have demonstrated that the second-harmonic wave of a diode laser (415 nm) could be used to determine polycyclic aromatic hydrocarbons.…”