Genotyping by Guanosine-Dependent Quenching
of Single-Labeled Fluorescein Probes
ANDREW O. CROCKETT*
Introduction
Fluorescent-labeled oligonucleotide probes can be used in real-time PCR for rapid detection of specific product and for fine sequence analysis [1-5]. Changes in
the magnitude of probe fluorescence upon hybridization to product DNA are
responsible for the versatility of fluorescent oligonucleotides in monitoring PCR.
Several techniques have been developed for using oligonucleotide probes to generate a fluorescent signal detectable by instruments such as the LightCycler. These
fluorescent-labeled molecules utilize the natural phenomenon of fluorescence
resonance energy transfer (FRET) to provide such a signal. During FRET, a donor
fluorophore absorbs light energy and transfers it to either an acceptor, a second
fluorophore that emits the light energy at a second, molecule-specific wavelength,
or to a quencher, a molecule that dissipates the energy as heat. Exonuclease
probes (TaqMan) [6], hairpin probes (Molecular Beacons) [7], and self-probing
primers (Scorpions) [8] are examples of FRET systems in which the fluorescence
of a donor is quenched by a second chromophore attached to the same molecule.
Because two fluorophores are conjugated to a single oligonucleotide, the design,
synthesis, and purification of these probes can be difficult and expensive.
Hybridization probe techniques eliminate many of the synthesis problems
inherent to dual-labeled probes by utilizing two single-labeled oligonucleotides,
one conjugated with the donor fluorophore and the other conjugated with an
acceptor [1-5, 9]. These oligonucleotides are designed to anneal to adjacent
regions of the PCR amplicon, bringing the attached fluorophores close enough
together for FRET. Because each probe only requires conjugation with a single fluorophore, synthesis and purification of hybridization probes is simpler than for
dual-labeled oligonucleotides. However, adjacent hybridization probes require a
greater region of hybridization to the template DNA which makes the design
prone to disruption by unexpected polymorphisms in the template. Also, signal
generation is dependent upon two separate hybridization events, as both probes
in a hybridization pair must anneal to a single template strand for the generation
of the FRET signal.
* Andrew o. Crockett (~) (e-mail: andrew.crockett@path.utah.edu)
Department of Pathology, University of Utah School of Medicine, 50 North Medical Drive,
Salt Lake City, UT 84132 USA
of Single-Labeled Fluorescein Probes
ANDREW O. CROCKETT*
Introduction
Fluorescent-labeled oligonucleotide probes can be used in real-time PCR for rapid detection of specific product and for fine sequence analysis [1-5]. Changes in
the magnitude of probe fluorescence upon hybridization to product DNA are
responsible for the versatility of fluorescent oligonucleotides in monitoring PCR.
Several techniques have been developed for using oligonucleotide probes to generate a fluorescent signal detectable by instruments such as the LightCycler. These
fluorescent-labeled molecules utilize the natural phenomenon of fluorescence
resonance energy transfer (FRET) to provide such a signal. During FRET, a donor
fluorophore absorbs light energy and transfers it to either an acceptor, a second
fluorophore that emits the light energy at a second, molecule-specific wavelength,
or to a quencher, a molecule that dissipates the energy as heat. Exonuclease
probes (TaqMan) [6], hairpin probes (Molecular Beacons) [7], and self-probing
primers (Scorpions) [8] are examples of FRET systems in which the fluorescence
of a donor is quenched by a second chromophore attached to the same molecule.
Because two fluorophores are conjugated to a single oligonucleotide, the design,
synthesis, and purification of these probes can be difficult and expensive.
Hybridization probe techniques eliminate many of the synthesis problems
inherent to dual-labeled probes by utilizing two single-labeled oligonucleotides,
one conjugated with the donor fluorophore and the other conjugated with an
acceptor [1-5, 9]. These oligonucleotides are designed to anneal to adjacent
regions of the PCR amplicon, bringing the attached fluorophores close enough
together for FRET. Because each probe only requires conjugation with a single fluorophore, synthesis and purification of hybridization probes is simpler than for
dual-labeled oligonucleotides. However, adjacent hybridization probes require a
greater region of hybridization to the template DNA which makes the design
prone to disruption by unexpected polymorphisms in the template. Also, signal
generation is dependent upon two separate hybridization events, as both probes
in a hybridization pair must anneal to a single template strand for the generation
of the FRET signal.
* Andrew o. Crockett (~) (e-mail: andrew.crockett@path.utah.edu)
Department of Pathology, University of Utah School of Medicine, 50 North Medical Drive,
Salt Lake City, UT 84132 USA