- Home
- IVD
- By Technology Types
- By Diseases Types
- By Product Types
- Research
- Resource
- Distributors
- Company
| Product Name | TaqMan SNP Genotyping Assay, Custom Design, 40x Primer-Probe Mix, FAM/VIC |
| Catalog No. | PRIPRO-0035 |
| Description | Custom-designed and pre-formulated TaqMan SNP genotyping assay delivered as a 40x concentrated primer-probe master mix ready for direct addition to qPCR reactions for allelic discrimination. For each single nucleotide polymorphism (SNP) of interest, the service includes bioinformatic design of two allele-specific TaqMan hydrolysis probes (6-FAM-labeled for Allele 1, VIC-labeled for Allele 2, both with BHQ-1 or non-fluorescent quencher at the 3' end) and a pair of locus-specific PCR primers flanking the polymorphic site. The 40x formulation is optimized by adjusting primer and probe concentrations and ratios to achieve robust fluorescent signal separation between homozygous reference, heterozygous, and homozygous variant genotype clusters in end-point allelic discrimination plots. Each assay includes a comprehensive design report with amplicon sequence, SNP position annotation, probe and primer information, predicted Tm values, and recommended thermal cycling protocol. The service supports human (hg19/hg38), mouse (mm10/mm39), rat (rn6/rn7), Arabidopsis, rice, maize, zebrafish, and custom genomes upon request. |
| Intended Use | High-throughput SNP genotyping by real-time PCR end-point allelic discrimination for: GWAS replication and fine-mapping studies; candidate gene association analysis; pharmacogenomic variant screening (CYP450 family, TPMT, UGT1A1, VKORC1, SLCO1B1); somatic mutation verification in oncology (BRAF V600E, KRAS, EGFR, JAK2 V617F); marker-assisted backcrossing and genomic selection in plant and animal breeding; transgenic zygosity testing; NGS-detected variant confirmation and segregation analysis. |
| Principle / Technology | The assay utilizes two allele-specific TaqMan probes with distinct fluorophores. The Allele 1 probe (FAM) is perfectly complementary to the reference allele at the SNP position; the Allele 2 probe (VIC) is perfectly complementary to the variant allele. A single-base mismatch at the SNP site dramatically reduces probe binding affinity (delta Tm typically 5-15 C), resulting in preferential cleavage of the perfectly matched probe by the 5'-exonuclease activity of Taq DNA polymerase. Post-PCR end-point fluorescence readings in FAM and VIC channels produce three distinct clusters on an allelic discrimination plot corresponding to homozygous Allele 1 (FAM-high, VIC-low), heterozygous (FAM-medium, VIC-medium), and homozygous Allele 2 (FAM-low, VIC-high) genotypes. |
| Detection Method | Customer provides: dbSNP rs ID (preferred), genomic coordinates with reference genome build, or 200-400 bp flanking sequence centered on the SNP. Our bioinformatics team designs probes (15-30 nt, Tm 65-70 C) and primers (18-25 nt, Tm 58-62 C) with amplicon size 75-150 bp. Synthesis: probes synthesized with 5'-FAM/BHQ-1 (Allele 1) and 5'-VIC/BHQ-1 (Allele 2); primers synthesized unmodified. 40x mix prepared by combining primers and probes at optimized ratios. Each assay batch is validated by genotyping a panel of 24-48 reference DNA samples with known genotypes. qPCR setup: add 0.5 uL 40x mix per 20 uL reaction; thermal cycling: 95 C 10 min, 40 cycles of 95 C 15 s, 60 C 60 s; end-point read in FAM and VIC channels. |
| Sample Type | Genomic DNA (1-20 ng per 20 uL reaction) extracted from blood, saliva, buccal swab, tissue, plant leaf, or cell culture. DNA quality: A260/A280 1.7-2.0, no PCR inhibitors. For pre-amplified DNA or whole-genome amplified DNA, dilute to 1-5 ng per reaction. |
| Performance Range / Specifications | Genotyping call rate: >98% on reference DNA panel; genotype concordance with sequencing: >99.5%; allelic discrimination cluster separation: >3 standard deviations between cluster centroids; amplicon size: 75-150 bp. |
| Sensitivity / LOD | Reliable genotyping from 0.5 ng genomic DNA per reaction (approximately 75 diploid human genome equivalents); detection of variant allele frequency as low as 5% for somatic mutation applications (with appropriate controls). |
| Specificity | Probe specificity: single mismatch at SNP site reduces probe binding Tm by 5-15 C, ensuring >95% discrimination between alleles; primer specificity: BLAST-verified unique amplicon in target genome; no cross-reactivity with pseudogenes or paralogs within 5 mismatches. |
| Reaction Conditions / Protocol | Reaction composition per 20 uL: 10 uL 2x qPCR master mix, 0.5 uL 40x assay mix, 20-50 ng DNA, water to 20 uL. Thermal cycling: 95 C 10 min, 40x (95 C 15 s, 60 C 60 s with data collection at 60 C), followed by end-point reading at 25 C or per instrument-specific protocol. |
| Components / Formulation | 40x TaqMan SNP Genotyping Assay Mix (primer and probe blend, 50 uL per assay — sufficient for 100 reactions at 20 uL volume with 0.5 uL 40x mix per reaction). Design Report (PDF) containing: reference SNP ID, genomic coordinates (build specified), amplicon sequence with SNP position highlighted, forward primer sequence and Tm, reverse primer sequence and Tm, Allele 1 probe sequence and dye/quencher, Allele 2 probe sequence and dye/quencher, recommended annealing/extension temperature, expected amplicon size, and BLAST specificity verification summary. |
| Storage Conditions | 40x Assay Mix at -20 C protected from light; stable for 24 months at -20 C; avoid repeated freeze-thaw cycles (aliquot upon receipt if needed). |
| Shelf Life | 24 months from date of synthesis at -20 C. |
| Package Specifications | Single assay (50 uL 40x mix, sufficient for 100 x 20 uL reactions), or bulk options for 500, 1,000, and 5,000 reactions. |
| Product Form | 40x concentrated liquid mix in TE buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA); colorless solution. |
| Quality Control | Each assay validated by: genotyping 24-48 human DNA samples with known genotypes (call rate >98%, concordance >99.5%); allelic discrimination plot inspected for three distinct well-separated clusters; no-template control (NTC) shows zero or negligible amplification in both channels; PCR efficiency 90-110% as determined by standard curve of 4 serial dilutions. |
| Key Features | Custom SNP genotyping assay with optimized 40x mix; dual-color allelic discrimination (FAM/VIC); >98% call rate; >99.5% concordance; includes comprehensive design report; 2-week turnaround from sequence submission to validated assay delivery. |
| Purity | HPLC-purified probes (>95% purity); desalted primers (>90% full-length); probe dye incorporation confirmed by UV-Vis spectrophotometry; no unlabeled oligonucleotides in probe preparations. |
| Concentration | 40x Assay Mix: typical final probe concentrations 8-10 uM each allele probe, primer concentrations 12-18 uM each (concentrations in 40x mix, before dilution to 1x). |
| Activity / Unit Definition | Allele discrimination: >95% probe cleavage specificity (ratio of FAM:VIC signal in homozygous samples >20:1 or <1:20). |
| Molecular Weight | Variable depending on probe and primer lengths; probes typically 7,000-11,000 Da; primers typically 5,500-8,000 Da. |
| Source / Origin | Oligonucleotides synthesized in-house by automated solid-phase phosphoramidite chemistry; 6-FAM, VIC, and BHQ-1 from commercial suppliers; all reagents nucleic acid-free and RNase-free grade. |
| pH Range / Optimal pH | Assay mix formulated in TE pH 8.0; compatible with qPCR reactions at pH 8.0-9.0; fluorescence pH dependent: optimal reading above pH 7.0. |
| Shipping Conditions | Ambient temperature (lyophilized or liquid); stable at ambient for up to 2 weeks transit. |
| Expiration Date / Stability | 24 months at -20 C; working dilution (1x): prepare fresh for each experiment; avoid freeze-thaw cycles of 40x stock. |
| Regulatory / Compliance | For research use only; not for diagnostic, clinical, or forensic use. Not intended for human identity testing or paternity determination. SNP assay content is the responsibility of the customer for off-target effects and intellectual property. |
| Compatibility | Compatible with universal qPCR master mixes without UNG (using dUTP); compatible with ABI QuantStudio, Bio-Rad CFX96/384/Opus, Roche LightCycler 480/96, Qiagen Rotor-Gene Q, Thermo PikoReal, and Analytik Jena qTOWER. Requires FAM/SYBR channel (Ex ~495 nm, Em ~520 nm) and VIC/HEX/JOE channel (Ex ~538 nm, Em ~554 nm). Not compatible with SYBR Green-based detection (spectral overlap with FAM). For instruments without VIC channel, HEX detection channel can be used with minor compensation adjustment. |
| Recommended Buffer System | 40x mix: TE buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA); compatible with all major commercial 2x qPCR master mixes. |
| Application Notes / Precautions | Centrifuge assay tube briefly before first use. For automated liquid handling, the 40x mix is compatible with all standard pipetting systems. Include at least 2 no-template controls (NTC) per 96-well plate. Include 3 positive control samples of known genotype (one of each genotype cluster if available) on each plate for allelic discrimination plot reference. For plates with multiple assays, use ROX or other passive reference dye where required by the instrument. Store unused 40x mix at -20 C protected from light; FAM and VIC are photostable for at least 12 months at -20 C in the dark. If genotyping call rate is <95%, check DNA quality (A260/A280, PCR inhibitor screen). |
| Batch-to-Batch Consistency | Genotyping call rate >98% on reference panel for every synthesis batch; cluster separation >3 SD between centroid means for every validated lot. |
For research use only, not for clinical use.
|
There is no product in your cart. |
Copyright © 2026 Alta DiagnoTech. All rights reserved.
