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    BiOligo

    qPCR Probes

    qPCR Probes

    Quantitative PCR (qPCR), also known as real-time PCR, is an essential tool in fundamental research and clinical diagnostics. It plays a vital role in detecting infectious diseases, cancer biomarkers, genetic disorders and other targets.

    We provide customized qPCR probes, including TaqMan probes, molecular beacon probes and super-quenched probes, all tailored to meet your specific research needs.

    Probe Design

    The selections of fluorophores and quenchers are essential to probe design:

    • An appropriate fluorophore choice is related to the qPCR instrument used;
    • An appropriate quencher choice is related to the quenching mechanism involved.
    Probe type Melting curve probe Super quench Taqman Molecular beacon
    RQ SQ BHQ BHQ-X TAMRA Eclipse Dabcyl
    / 430- NIR 430 nm- NIR 390-625 nm 520-600 nm 390-625 nm 380-530 nm
    Tm Enhancement N N N Increasing by 10 degrees or more N N N
    A-T Rich Sequence Compatibility Y Y N Y N N N
    Background fluorescence None None None None Yes None None
    Sensitivity +++ +++++ +++ ++++ ++ +++ +
    Specificity +++ +++ + +++ + + ++
    Design Complexity + + + ++ + + +++
    Synthesis Complexity + + + +++ + + ++

     

    Common Fluorophore and Quencher Combinations

    General  qPCR Probes

    BHQ Probes

    The BHQ quenchers include BHQ-0 (430–520 nm), BHQ-1 (480–580 nm), BHQ-2 (550–650 nm), and BHQ-3 (620–730 nm). These non-fluorescent quenchers efficiently absorb fluorescence emitted by reporter dyes within their respective spectral ranges. With a broad absorption spectrum spanning from 430 nm to the near-infrared region, BHQ quenchers provide strong quenching efficiency, effectively reducing background noise and thereby enhancing the signal-to-noise ratio. Ideal for multiplex qPCR applications.

    BHQ-X Probe

    BHQ-X probes are optimized versions of traditional TaqMan probes. They feature a 3' end conjugated with BHQ-X, a minor groove binder that increases the probe’s melting temperature (by approximately 10 °C) and stabilizes hybridization with the target. This allows BHQ-X probes shorter than conventional probes while offering improved specificity. Additionally, BHQ-X probes incorporate a non-fluorescent quencher (NFQ) that effectively quenches the signal from the reporter dye at the 5' end, reducing background fluorescence and enhancing detection sensitivity. Recommended for SNP detection.

    TAMRA Probe

    TAMRA was one of the first quenchers used in TaqMan probe technology. Although it was widely used in early designs, its fluorescent properties result in higher background signals and limited spectral compatibility, making it less suitable for qPCR applications.

    Molecular Beacon Probe

    Molecular beacons are probes capable of forming a stem-loop (hairpin) structure. The 5′ end is labeled with a fluorescent reporter and a quencher (typically Dabcyl) at the 3′ end. The loop region is complementary to the target sequence, while the stem is formed by complementary sequences at both ends of the probe. During the annealing process, the probe hybridizes with the target, separating the fluorophore from the quencher and generating a fluorescent signal. The unique hairpin design makes molecular beacons exceptionally specific in distinguishing target sequences, ideal for real-time PCR, SNP detection, and mutation analysis.

     

    Super-quenched qPCR Probes

    To reduce background fluorescence caused by incomplete quenching in traditional TaqMan probes, We developed the Super-Quenched (SQ) probe. By adding an extra internal quencher and a unique 3' quencher, this design effectively lowers background noise, improves sensitivity, and prevents fluorescence leakage.

    Features

    • High Sensitivity | LOD to 1 copy

    • High Signal-to-Noise Ratio (SNR) | Effectively reduces background fluorescence, increase endpoint fluorescence signal intensity

    • Ideal for Multiple qPCR Detection | Minimizes fluorescence crosstalk, minimize non-specific amplification

    • Applicable to AT-rich Targets

    Recommended Combinations

    Cases

    Comparison of Modified Fluorescence for Different Quenchers in the Same Sequence.

    qPCR Applicable —— Lower background signal and higher SNR

    Channel
    Target
    		 Figure
    FAM
    AsFV-p72
    HEX
    Monkeypox plasmid
    Cy5
    COVID-19

     

    Melt Curve qPCR Probes (RQp)

    To address the limitations of conventional multicolor melting curve analysis (low sensitivity, uneven baselines, inverted peaks, and poor compatibility with mutation-prone templates), we have developed RQp probes for melting curve applications.
    This probe allows flexible Tm adjustment without additional quencher. It is compatible with common qPCR channels (FAM, VIC, ROX, Cy5) and supports single-tube multiplex detection, including multiplex detection of pathogenic DNA and RNA.


    Features

    Flexible Probe Design
    • Unlike FRET probes, which are limited in length (typically 15–30 bp), or molecular beacons that require complex secondary structures, RQp probes have no strict length constraints.
    • Tm values can be easily adjusted through simple sequence design, significantly reducing design workload.
    Stable Curves with Flat Baselines
    • RQp probes do not generate fluorescence changes during amplification or melting curve analysis. This ensures a flat and clean baseline, minimizing the risk of inverted peaks and enabling more accurate interpretation.
    Eliminates Nonspecific Melt Peaks
    • With proprietary fluorescent modifications, RQp probes effectively avoid nonspecific melting peaks, improving result clarity and reducing the risk of false positives.

    Recommended Combinations

    Channel Modification
    5’- 3’-
    FAM BF490 RQP
    VIC/HEX BF533
    ROX BF590
    Cy5 BF648
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