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Fluorescent nucleic acid probes, which use energy transfer, include such constructs as molecular beacons, molecular break lights, Scorpion primers, TaqMan probes, and others. These probes signal detection of their targets by changing either the intensity or the color of their fluorescence. Not surpr- ingly, these luminous, multicolored probes carry more flashy names than their counterparts in the other fields of molecular biology. In recent years, fluor- cent probes and assays, which make use of energy transfer, have multiplied at a high rate and have found numerous applications. However, in spite of this explosive growth in the field, there are no manuals summarizing different p- tocols and fluorescent probe designs. In view of this, the main objective of Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols is to provide such a collection. Oligonucleotides with one or several chromophore tags can form fluor- cent probes capable of energy transfer. Energy transport within the probe can occur via the resonance energy transfer mechanism, also called Förster tra- fer, or by non-Förster transfer mechanisms. Although the probes using Förster transfer were developed and used first, the later non-Förster-based probes, such as molecular beacons, now represent an attractive and widely used option. The term “fluorescent energy transfer probes” in the title of this book covers both Förster-based fluorescence resonance energy transfer (FRET) probes and probes using non-FRET mechanisms. Energy transfer probes serve as molecule-size sensors, changing their fluorescence upon detection of various DNA reactions.
Fluorescent nucleic acid probes, which use energy transfer, include such constructs as molecular beacons, molecular break lights, Scorpion primers, TaqMan probes, and others. These probes signal detection of their targets by changing either the intensity or the color of their fluorescence. Not surpr- ingly, these luminous, multicolored probes carry more flashy names than their counterparts in the other fields of molecular biology. In recent years, fluor- cent probes and assays, which make use of energy transfer, have multiplied at a high rate and have found numerous applications. However, in spite of this explosive growth in the field, there are no manuals summarizing different p- tocols and fluorescent probe designs. In view of this, the main objective of Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols is to provide such a collection. Oligonucleotides with one or several chromophore tags can form fluor- cent probes capable of energy transfer. Energy transport within the probe can occur via the resonance energy transfer mechanism, also called Förster tra- fer, or by non-Förster transfer mechanisms. Although the probes using Förster transfer were developed and used first, the later non-Förster-based probes, such as molecular beacons, now represent an attractive and widely used option. The term “fluorescent energy transfer probes” in the title of this book covers both Förster-based fluorescence resonance energy transfer (FRET) probes and probes using non-FRET mechanisms. Energy transfer probes serve as molecule-size sensors, changing their fluorescence upon detection of various DNA reactions.
Research toward a multiplexed nucleic acid biosensor that uses quantum dots (QDs) as donors in a fluorescence resonance energy transfer (FRET) assay is described. Optical fibers were modified with mixed films composed of different colours of QDs and different oligonucleotide probes that served as scaffolds for the hybridization of the corresponding target nucleic acid sequences. Fluorescent dyes that were suitable as acceptors for each QD donor were associated with hybridization and provided an analytical signal through FRET-sensitized emission. Different detection channels were achieved through the combination of different donors and acceptors: green emitting QDs with Cyanine 3 or Rhodamine Red-X; and red emitting QDs with Alexa Fluor 647. A detection channel that used the direct excitation of Pacific Blue complemented the FRET pairs. One-plex, two-plex, three-plex and four-plex hybridization assays were demonstrated. A sandwich assay format was adopted to avoid target labeling. Detection limits were 1-10 nM (1-12 pmol) and analysis times were 1-4 h. Single nucleotide polymorphisms were discriminated in multiplexed assays, and the potential for reusability was also demonstrated. Non-selective interactions between QDs and oligonucleotides were characterized, and routes toward the optimization of the QD-FRET hybridization assays were identified. A basic model for multiple FRET pathways in a mixed film was also developed. In addition to the advantages of solid-phase assays, the combination of QDs and FRET was advantageous because it permitted multiplexed detection using a single excitation source and a single substrate, in the ensemble, and via ratiometric signals. Spatial registration or sorting methods, imaging or spatial scanning, and single molecule spectroscopy were not required. The research in this thesis is expected to enable new chip-based biosensors in the future, and is an original contribution to both bioanalytical spectroscopy and the bioanalytical applications of nanomaterials.
The use of fluorescent and luminescent probes to measure biological function has increased dramatically since publication of the First Edition due to their improved speed, safety, and power of analytical approach. This eagerly awaited Second Edition, also edited by Bill Mason, contains 19 new chapters and over two thirds new material, and is a must for all life scientists using optical probes. The contents include discussion of new optical methodologies for detection of proteins, DNA and other molecules, as well as probes for ions, receptors, cellular components, and gene expression. Emerging and advanced technologies for probe detection such as confocal laser scanning microscopy are also covered. This book will be essential for those embarking on work in the field or using new methods to enhance their research. TOPICS COVERED: * Single and multiphoton confocal microscopy * Applications of green fluorescent protein and chemiluminescent reporters to gene expression studies * Applications of new optical probes for imaging proteins in gels * Probes and detection technologies for imaging membrane potential in live cells * Use of optical probes to detect microorganisms * Raman and confocal raman microspectroscopy * Fluorescence lifetime imaging microscopy * Digital CCD cameras and their application in biological microscopy
The unique optical properties of quantum dots (QDs) are of interest in the development of nucleic acid diagnostics. The potential for a simultaneous two-colour diagnostic scheme for nucleic acids operating on the basis of fluorescence resonance energy transfer (FRET) has been demonstrated. Upon ultraviolet excitation, two-colours of CdSe/ZnS quantum dots with conjugated oligonucleotide probes acted as energy donors yielding FRET-sensitized acceptor emission upon hybridization with fluorophore labeled target oligonucleotides. The use of an intercalating dye to improve signal-to-noise was also demonstrated. The major limitation of the system was the non-specific adsorption of oligonucleotides, which was characterized extensively. Adsorptive interactions were found to affect the conformation of oligonucleotides conjugated to QDs, the kinetics of hybridization with QD-DNA conjugates, and the thermal stability of those hybrids. In addition, it was found that thiol-alkyl-acid capped QDs exhibited pKa correlated ligand-chromism and radiative decay rate-driven changes in quantum yield.
Thiazole orange (TO) with different tethers were synthesized to be attached to oligonucleotides. The FRET of TO in solution with double-stranded DNA (dsDNA) was investigated with BlackHole (BHQ1) or ((4-dimethylamino)phenyl)azo)benzoic acid (DABCYL) quenchers which decreased the fluorescence 2.9 +/- 7% and 2.5 +/- 10% times, respectively. A quenching mechanism could therefore be designed to transduce hybridization. The FRET of N, N, N, N-tetramethylcarboxyrhodamine (TAMRA) and IowaBlackRQ RTM (IABLK) linked to complementary oligonucleotides immobilized on glass substrates was investigated; IABLK quenched TAMRA fluorescence. However, surface bound dsDNA caused some self-quenching of TAMRA. Solution FRET using TAMRA/IABLK at 24.5°C and 60°C with complementary and mismatched DNA was measured to investigate potential for mismatch detection. The probe sequence was based on the determinate for Spinal Muscular Atrophy (SMA). Signal intensity differed between complementary and the mismatch samples at 60°C, indicating mismatch detection potential. Results suggest the possibility of designing tethered fluorophore-quencher pairs for transduction of hybridization for development of optical nucleic acid biosensors for SMA screening.
The unique spectroscopic properties of quantum dots (QDs) are of interest for application in intracellular studies of gene expression. QDs derivatized with single-stranded probe oligonucleotides were used to detect complementary target sequences via hybridization and fluorescence resonance energy transfer (FRET). As nucleic acid targets are not labeled within cells, a displacement assay for nucleic acid detection featuring QDs as FRET donors was developed. QDs conjugated with oligonucleotide probes and then pre-hybridized with labeled target yielded efficient FRET in vitro. Studies in vitro confirmed that displacement kinetics of pre-hybridized target was a function of the stability of the initial hybridized complex. Displacement was observed as reduction in FRET intensity coupled with regeneration of QD fluorescence. By engineering the sequence of the labeled target, faster displacement was possible. The QD-probe+target system was successfully delivered into cells via transfection. Although QDs with their cargo remained sequestered in endosomal vesicles, fluorescent properties were retained.