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

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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
  • Requires 10-100 ng cfDNA from blood plasma, or 10-1000 ng DNA from tissue biopsy
  • Requires 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

List of Detected and Quantitated Mutations

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

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

Compatible Instruments

qPCR

Sanger sequencing

Background

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.

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