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| Product Name | Dual-Labeled Hydrolysis Probe (FAM-BHQ1) for qPCR |
| Catalog No. | PRIPRO-0010 |
| Description | A dual-labeled fluorescent hydrolysis probe for quantitative real-time PCR (qPCR), featuring a 5′-FAM (6-carboxyfluorescein) fluorophore and a 3′-Black Hole Quencher 1 (BHQ1) moiety. The probe operates on the principle of fluorescence resonance energy transfer (FRET): when the probe is intact, the proximity of BHQ1 to FAM quenches fluorescence via both FRET and static quenching mechanisms. During PCR, the 5′→3′ exonuclease activity of Taq DNA polymerase cleaves the probe, separating the fluorophore from the quencher and generating a fluorescent signal proportional to target amplification. The probe is HPLC purified to >95% dual-labeled purity. Sequence is custom-designed by the user and synthesized with each batch verified by mass spectrometry. |
| Intended Use | Designed for quantitative real-time PCR detection of specific DNA or cDNA targets, including gene expression analysis, pathogen detection, SNP genotyping by allele-specific probe cleavage, and copy number variation analysis. |
| Principle / Technology | FRET-based quenching: 5′ FAM reporter fluorescence is quenched by 3′ BHQ1 dark quencher until probe hydrolysis by Taq polymerase 5′→3′ exonuclease activity during PCR extension |
| Detection Method | Real-time fluorescence monitoring in FAM channel (Ex 495 nm / Em 520 nm); signal increase correlates with target template copy number |
| Sample Type | Purified DNA or cDNA from cells, tissues, blood, swabs, or environmental sources |
| Performance Range / Specifications | Probe length 18–30 bases; Tm 65–70 °C (5–10 °C above primer Tm); GC content 40–60%; no 5′-G or runs of identical bases |
| Sensitivity / LOD | Capable of detecting fewer than 10 copies of target template per reaction; linear dynamic range of 6–7 orders of magnitude |
| Specificity | Sequence-specific detection; probe binding and cleavage require perfect complementarity; single-base mismatches significantly reduce cleavage efficiency, enabling allele discrimination |
| Reaction Conditions / Protocol | Use at 100–250 nM final probe concentration in qPCR reaction with 300–900 nM each primer. Standard two-step cycling: 95 °C for 15–30 s denaturation, 60 °C for 30–60 s annealing/extension with fluorescence acquisition. |
| Components / Formulation | Single tube containing lyophilized dual-labeled HPLC-purified FAM-BHQ1 probe; certificate of analysis |
| Storage Conditions | –20 °C in the dark; light-sensitive; use amber tubes for working solutions |
| Shelf Life | 12 months at –20 °C dry (dark); 6 months in solution at –20 °C (dark) |
| Package Specifications | Foil-wrapped tube with 5–50 nmol scale; custom scales available |
| Product Form | Lyophilized pellet (dual-labeled); pale yellow to colorless |
| Quality Control | HPLC purity ≥95% dual-labeled (both dye and quencher confirmed); MALDI-TOF MS; quenching efficiency verified by fluorescence measurement before and after DNase I digestion; endotoxin-free available upon request. |
| Key Features | BHQ1 dark quencher — no native fluorescence, enabling lower background; FRET and static quenching mechanism for efficient signal suppression; HPLC purified to >95%; sequence verified by MS; compatible with all FAM-channel qPCR instruments. |
| Purity | ≥95% dual-labeled by HPLC; residual unlabeled or single-labeled oligo <5% |
| Concentration | Lyophilized; resuspend to 100 µM stock based on A260 measurement; typical yield 15–40 OD260 at 25 nmol scale |
| Activity / Unit Definition | Quenching efficiency >95%; fluorescence increase >10-fold upon complete DNase I digestion; signal-to-noise ratio typically >50:1 |
| Molecular Weight | Calculated from probe sequence plus ~537 Da (FAM) and ~547 Da (BHQ1) modifications; reported on COA |
| Source / Origin | Synthetic oligonucleotide; FAM and BHQ1 phosphoramidites from chemical synthesis |
| pH Range / Optimal pH | Optimal performance in qPCR buffer pH 8.0–8.5; probe stable pH 6.0–9.0 |
| Shipping Conditions | Ambient temperature in foil-wrapped light-protective container |
| Expiration Date / Stability | 12 months at –20 °C dry (dark); 6 months in nuclease-free water or TE at –20 °C (dark); avoid repeated freeze-thaw |
| Regulatory / Compliance | For research use only; not for diagnostic applications without validation |
| Compatibility | Compatible with all qPCR master mixes having FAM channel detection: ABI 7500/7900HT/QuantStudio, Bio-Rad CFX96/CFX384, Roche LightCycler 480/96, Qiagen Rotor-Gene Q, and others |
| Recommended Buffer System | Standard qPCR buffer (Tris-HCl pH 8.0–8.3, KCl, MgCl₂ 3–5 mM, dNTPs, Taq polymerase); probe stable in these conditions |
| Application Notes / Precautions | Design probe with Tm 5–10 °C above primer Tm. Avoid 5′-G (quenches FAM). Keep working stock in amber tube. For multiplex qPCR, verify no cross-reactivity between probes before running full panel. |
| Batch-to-Batch Consistency | Batch-to-batch quenching efficiency within ±5%; HPLC purity maintained within ±2%; MS mass accuracy ±0.05% |
For research use only, not for clinical use.
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