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| Product Name | Dual-Labeled BHQ-1 qPCR Probes, 6-FAM, HPLC Purified, Custom Sequence |
| Catalog No. | PRIPRO-0034 |
| Description | Custom-synthesized dual-labeled hydrolysis (TaqMan) probes with 6-carboxyfluorescein (6-FAM) as the 5'-fluorophore and Black Hole Quencher 1 (BHQ-1) as the 3'-quencher, purified by reversed-phase HPLC for the highest level of purity. Each probe is synthesized according to the customer-provided target sequence (18-30 bases), with 6-FAM covalently attached to the 5'-terminus via a C6-amino linker and BHQ-1 conjugated to the 3'-terminus via a controlled-pore glass (CPG) solid support during automated synthesis. These probes are designed for real-time quantitative PCR (qPCR) detection of specific DNA or cDNA targets, operating via FRET-based (Fluorescence Resonance Energy Transfer) quenching: in the intact probe, BHQ-1 quenches 6-FAM fluorescence through non-fluorescent energy transfer. During PCR, Taq DNA polymerase's 5'->3' exonuclease activity cleaves the probe, separating the fluorophore from the quencher, resulting in fluorescence increase proportional to target amplification. |
| Intended Use | Real-time quantitative PCR (qPCR) detection and quantification of specific DNA or cDNA targets in gene expression analysis, pathogen detection, SNP genotyping (when designed across SNP site), copy number variation analysis, and multiplex qPCR assays (combining 6-FAM probes with other fluorophore-quencher combinations for multi-target detection). |
| Principle / Technology | Hydrolysis probe (TaqMan) principle: 5'-exonuclease activity of Taq or similar DNA polymerase cleaves the hybridized probe during primer extension. Cleavage physically separates 6-FAM (donor) from BHQ-1 (acceptor/quencher), eliminating FRET and resulting in increased 6-FAM fluorescence (Ex/Em = 495/520 nm) that accumulates with each PCR cycle. BHQ-1 is a dark quencher (no native fluorescence), absorbing broadly from 480-580 nm with absorption maximum at 534 nm, providing excellent quenching of 6-FAM (emission 520 nm). |
| Detection Method | Customer submits DNA target sequence; Alta DiagnoTech designs probe sequence (18-30 bases, Tm 65-70 C, GC content 40-60%, no G at 5' end, no runs of >3 identical bases); probe synthesized on automated DNA synthesizer using standard phosphoramidite chemistry with 6-FAM phosphoramidite and BHQ-1 CPG; probe cleaved, deprotected, and purified by RP-HPLC to single peak; quality control by MALDI-TOF mass spectrometry and capillary electrophoresis; probe delivered lyophilized or in TE buffer as specified. |
| Sample Type | Custom DNA sequence provided by customer as plain text (5' to 3'); recommended length 60-150 bp target amplicon for optimal qPCR efficiency. |
| Performance Range / Specifications | Probe length: 18-30 nucleotides; 5' modification: 6-FAM (Ex/Em 495/520 nm); 3' modification: BHQ-1 (absorption 480-580 nm, max 534 nm); Tm: designed at 65-70 C (probe Tm should be 5-10 C higher than primer Tm); purity: >=95% by HPLC; extinction coefficient: calculated from sequence composition; molecular weight: confirmed by MALDI-TOF MS (within 0.1% of calculated). |
| Sensitivity / LOD | Fluorescent signal detectable with as few as 10^9 molecules of cleaved probe (typical qPCR instrument sensitivity); probe capable of detecting 5-10 copies of target DNA per qPCR reaction when used with optimized primers and amplification conditions. |
| Specificity | HPLC purification removes truncated (n-1, n-2) and failure sequences that contribute to background fluorescence and non-specific signal; dual-labeled (5' FAM, 3' BHQ-1) confirmed by absorbance spectroscopy (A260/A495 ratio); MALDI-TOF MS confirms correct molecular weight; probe sequence specificity determined by user's bioinformatic design. |
| Reaction Conditions / Protocol | Probe working concentration: 100-250 nM final in qPCR reaction; thermal cycling standard: 95 C 10 min (polymerase activation), 40 cycles of 95 C 15 s, 60 C 60 s (combined annealing/extension with fluorescence acquisition at annealing/extension step). |
| Components / Formulation | Customer receives: Custom dual-labeled probe, lyophilized (1-10 nmol as ordered) or in TE buffer (100 uM), ship at ambient temperature; Specification Sheet with: probe sequence, Tm, MW (calculated and MS-confirmed), extinction coefficient, HPLC purity chromatogram, MALDI-TOF mass spectrum. |
| Storage Conditions | Lyophilized probe: stable at -20 C for 24 months; probe in TE (100 uM): stable at -20 C for 12 months; avoid repeated freeze-thaw cycles (>10); protect from light (aluminum foil wrap or amber tube); working dilution (10 uM): stable at 2-8 C for 3 months. |
| Shelf Life | 24 months from date of synthesis at -20 C. |
| Package Specifications | 1 nmol, 2 nmol, 5 nmol, 10 nmol (lyophilized); or 100 uM in TE buffer (volume equivalent to ordered amount). |
| Product Form | Lyophilized pellet (sodium salt) or 100 uM solution in TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA); appearance: colorless to pale pink (lyophilized) or colorless solution. |
| Quality Control | Each probe quality controlled by: (1) RP-HPLC (purity >=95%, single major peak >=85% total area); (2) MALDI-TOF mass spectrometry (observed MW within 0.1% of calculated); (3) UV-Vis spectrophotometry (A260 for quantitation, A495 for 6-FAM, A534 for BHQ-1); (4) Capillary electrophoresis (single peak, purity >=95%); (5) Fluorescence quenching efficiency (>=95% quenching in intact probe, >10-fold fluorescence increase after DNase I digestion). |
| Key Features | 6-FAM/BHQ-1 dye pair (most widely used qPCR combination); HPLC purified to >=95%; MALDI-TOF mass confirmation; custom sequence (user-specified); CE compliant; RNase-free; standard 5-7 business day synthesis and QC turnaround. |
| Purity | >=95% by RP-HPLC; single peak >=85% area; full-length product confirmed by mass spectrometry; free dye <1%. |
| Concentration | Lyophilized: specified nmol per tube (typically 1-10 nmol); 100 uM solution for liquid format; working concentration 100-250 nM in qPCR. |
| Activity / Unit Definition | Fluorescence quenching efficiency >=95% in intact probe (FAM intensity <5% of unquenched FAM standard at same concentration); upon DNase I digestion: >10-fold fluorescence increase. |
| Molecular Weight | Calculated based on user-specified sequence; typically 7,000-11,000 Da for 20-30 mer dual-labeled probe; confirmed by MALDI-TOF MS. |
| Source / Origin | Synthesized in-house by automated solid-phase phosphoramidite synthesis; 6-FAM phosphoramidite and BHQ-1 CPG from commercial suppliers; all reagents RNase-free grade. |
| pH Range / Optimal pH | Probe stable at pH 6.0-9.0; FAM fluorescence pH dependent (pKa ~6.4, maximal fluorescence above pH 7.0); qPCR reactions typically at pH 8.0-8.5. |
| Shipping Conditions | Ambient temperature (lyophilized); cold pack optional for liquid format; probe stable at ambient for 4 weeks lyophilized. |
| Expiration Date / Stability | 24 months at -20 C lyophilized; 12 months at -20 C in TE (100 uM); working dilution (10 uM): 3 months at 2-8 C protected from light. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. Custom synthesis service — probe sequence is the responsibility of the customer for specificity, off-target hybridization, and intellectual property considerations. |
| Compatibility | Compatible with all commercially available qPCR master mixes (SYBR Green master mixes are NOT compatible — use probe-based master mixes without SYBR Green). Compatible with AB QuantStudio, Bio-Rad CFX, Roche LightCycler, Qiagen Rotor-Gene, and other real-time PCR instruments with FAM/SYBR channel (Ex 470-500 nm, Em 510-540 nm). Do not use in SYBR Green detection channel simultaneously (spectral overlap). For multiplex qPCR, pair 6-FAM/BHQ-1 probes with VIC/BHQ-1, NED/BHQ-2, or Cy5/BHQ-3 probes for multi-channel detection. |
| Recommended Buffer System | Reconstitute in TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) or nuclease-free water; avoid using DEPC-treated water (DEPC may degrade probes over extended storage). |
| Application Notes / Precautions | Upon receipt, briefly centrifuge tube before opening to collect lyophilized pellet at bottom. Reconstitute in TE buffer to 100 uM stock (e.g., for 5 nmol: add 50 uL TE). Determine actual concentration by measuring A260 on NanoDrop (1 A260 unit = 33 ug/mL ssDNA). Prepare 10 uM working dilution in TE and store at -20 C protected from light. Avoid exposure to light (FAM photobleaches). For multiplex assay design, ensure probe Tm is 5-10 C higher than primer Tm. The 5' base of the probe must NOT be G (guanine quenches FAM even after cleavage). Submit sequence in standard IUPAC nomenclature (A, C, G, T only). |
| Batch-to-Batch Consistency | HPLC purity >=95% for every synthesis; MALDI-TOF mass within 0.1% of calculated; fluorescence quenching efficiency >=95%. |
For research use only, not for clinical use.
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