VarTrace™ BRAF Assay
For research use only. Not for use in diagnostic procedures.
BRAF gene is frequently mutated in specific cancers, including melanoma, papillary thyroid cancer, and colon cancer. The most prevalent BRAF mutations detected in these cancer types are missense mutations that introduce an amino acid substitution at valine 600. The presence of these specific mutations can guide treatment with targeted therapies such as TAFINLAR® (dabrafenib), MEKINIST®(trametinib), and ZELBORAF®(vemurafenib).
The VarTrace™ BRAF Assay enables highly sensitive detection and quantification of BRAF mutations for cancer patients. 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.
Limit of detection down to 0.1% VAF
Able to quantify mutation VAF in original sample
Applicable to a variety of sample types
Wide range of mutations covered
- Detects any mutations in codons 596–601 (c. 1786-1802)
- Compatible with plasma samples, tumor biopsies, paraffin-embedded sections (FFPE), and fresh frozen tumors
- Requires 10-100 ng cfDNA from blood plasma, or 10-1,000 ng DNA from tissue biopsy
- Delivers results in 8 hours, with 2.5 hours of hands-on time
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.
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)
Step 3: Sanger Sequencing
BDA products are prepared for Sanger sequencing, including PCR product purification, cycle sequencing and sequencing clean-up.
3.5 hrs (1hr hands-on)
Purified Sanger products are analyzed by capillary electrophoresis.
2 hrs (15 min hands-on)