Time to read: 2 minute read
Updated : Thu, June 1, 2023 @ 10:49 AM
Originally published : Wed, Feb 24, 2010 @ 12:20 PM
Updated : Thu, June 1, 2023 @ 10:49 AM
Originally published : Wed, Feb 24, 2010 @ 12:20 PM
A BHQ® probe is a dual-labeled oligonucleotide covalently labeled with a fluorophore and a Black Hole Quencher® (BHQ) dye.
Q. What are the main components of a dual-labeled Black Hole Quencher® (BHQ) probe?
Q. What is its native conformation at melting and annealing temperatures?
Melted - random coil conformation
Unhybridized - unrestricted hairpin, FRET-quenched (no signal)
Hybridized- stable double helix, probe-target hybrid (signal)
Q. How does it yield signal?
When a complementary sequence is available, the probe hybridizes to the complementary sequence. Conformational changes associated with hybridization separate the fluorophore and quencher, decreasing FRET quenching and releasing fluorescence.
Q. What happens with each consecutive PCR cycle?
1. Heat melts or denatures the probe, sense and antisense strands of a DNA duplex.
2. As temperatures cool, hydrophobicity and electrostatics promote dye-dye attractions and enhance fluorescent quenching.
3. At annealing temperatures, the primers and the BHQ probe anneal to their complementary sequences within the target DNA. Conformational changes during hybridization separate the dyes which decreases FRET quenching thus releasing fluorescence.
4. During elongation, the DNA polymerase incorporates nucleotides complementary to the strand as it progresses in a 5' to 3' direction from the primer. When the polymerase encounters the 5'-end of the probe, it cleaves off the nucleotide, or a flap of nucleotides, with the bound reporter dye, thereby permanently separating the reporter and quencher dyes.
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Dual-labeled BHQ probes have replaced earlier reporter-quencher dye pairings, such as FAM-TAMRA or FAM-DABCYL. In such sub-optimal probes, the quencher has inherent limitations such as auto-fluorescence or insufficient quenching at certain wavelengths which limit the choice of quenchable fluorophores. In contrast, the BHQ dyes:
BHQ dyes can be paired with all common reporter dyes emitting between the ultraviolet and infrared wavelengths, thereby making multiplexed hybridisation assays easy to design and interpret.
Applications: Single and multiplex, quantitative and qualitative, real-time and endpoint PCR analyses; allelic discrimination; and SNP detection.
Written by: Christina Ferrell, Ph.D., Technical Applications Specialist
LGC, Biosearch Technologies is the complete Genomics portfolio from LGC. Providing genomic analysis tools, instrumentation and services to the genomic scientific discovery sector worldwide, with focus on across ag bio, pharma and molecular diagnostics. Visit our home page to view our products and services.
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