Key Features
Ultrasensitive
Limit of detection down to 0.1% VAF
Comprehensive
Wide range of mutations covered
Quantitative
Able to quantitate mutation VAF in original sample
Sample flexibility
Applicable to a variety of sample types
Benefits
- Detects 400 EGFR mutations and quantitates 43 mutations
- Enables distinction of cis/trans mutations in C797S/T790M
- Compatible with plasma samples, tumor biopsies, paraffin-embedded sections (FFPE), and fresh frozen tumors
- Accepts 10-100 ng cfDNA from blood plasma, or 10-1000 ng DNA from tissue biopsy
- Accepts 2-10 ml of blood, 1-5ml of blood plasma, as little as 1mm*1mm (5 μm) tissue sections (fresh frozen or FFPE)
- Delivers results in 8 hours, with 2.5 hours of hands-on time
Sample Data
- Variant verification by Sanger sequencing
- Because NuProbe’s BDA technology enriches the variant allele frequency (VAF) of mutations by over 200-fold, Sanger’s limit of detection of 20% VAF is effectively improved to 0.1% VAF. Sanger sequencing can be applied to confirm the identity of quantities detected and quantitated by qPCR using standard sequencing protocols.
- Variant verification by Sanger sequencing
- Because NuProbe’s BDA technology enriches the variant allele frequency (VAF) of mutations by over 200-fold, Sanger’s limit of detection of 20% VAF is effectively improved to 0.1% VAF. Sanger sequencing can be applied to confirm the identity of quantities detected and quantitated by qPCR using standard sequencing protocols.
Workflow
Step 1: DNA Extraction
DNA is extracted from clinical samples with commercial extraction kits such as cobas® cfDNA Sample Preparation Kit, QIAamp® DNA Blood Mini Kit, GeneRead® DNA FFPE Kit.
40 min (40 min hands-on)
Step 2: qPCR BDA Reaction
NuProbe’s qPCR/Sanger kit reagents and clinical samples are prepared for qPCR reactions. After running the reaction, Ct results and BDA products are obtained.
1.5 hrs (30 min hands-on)
2.5 hrs total time for qPCR reactions (STEP1 + STEP2)
Step 3: Sanger Sequencing (Optional)
BDA products are prepared for Sanger sequencing, including PCR product purification, cycle sequencing and sequencing clean-up. Purified Sanger products are analyzed by capillary electrophoresis.
5.5 hrs (1.5 hrs hands-on)
8 hrs total time including Sanger Sequencing (STEP1+STEP2+STEP3)
Compatible Instruments
qPCR
BioRad CFX96
ABI 7500
Cobas z480
Sanger sequencing
ABI 3500 Dx Genetic Analyzer
Background
Catalog number
1R-0201-S
24 reactions
For research use only. Not for use in diagnostic procedures.
EGFR mutations are present in 10-30% non-small cell lung cancer (NSCLC) patients. Specific mutations can guide treatment with targeted therapies such as TARCEVA® (erlotinib), TAGRISSO® (osimertinib), IRESSA® (gefitinib).
The VarTrace™ EGFR Assay enables highly sensitive detection and quantification of EGFR mutations for patients with advanced or metastatic NSCLC. The kit uses NuProbe’s PCR-based Blocker Displacement Amplification (BDA) technology to enable the selective amplification of low abundant sequence variants (SNV and indels) in a background of wildtype DNA. Following PCR enrichment, Sanger sequencing is applied to reveal the identity of the variants.
