Key Features

Sensitive

Limit of detection down to 0.1% VAF

Quantitative

Able to quantify mutation VAF in original sample

Versatile

Applicable to a variety of sample types

Comprehensive

Wide range of mutations covered

User-friendly

Easy workflow

For research use only. Not for use in diagnostic procedures.

The VarTrace products enable highly sensitive detection and quantification of mutations for NSCLC, AML, melanoma patients. The kits use 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.

Product Offerings

EGFRMutation positionMutation typeDetectable mutations
Hotspot mutations in Exon 18-21*SNV, Indel272

BRAFMutation positionMutation typeDetectable mutations
Any mutations in codons 596–601SNV, Indel67

AMLMutation position (AA)Mutation typeDetectable mutations
FLT3 (TKD)835-836SNV45
DNMT3A882SNV17
IDH1132SNV17
IDH2140, 172SNV31
KIT816SNV36
NPM1288Indel105

Product Specifications

Kit sizeEGFR, AML: 24 reactions / kit
BRAF: 33 reactions / kit
Turnaround timeSame day results
Sample typePlasma samples, tumor biopsies, paraffin-embedded sections (FFPE), and fresh frozen tumors (only blood samples for AML panels)
Sample input10-100 ng cfDNA from blood plasma, or 10-1000 ng DNA from tissue biopsy or blood plasma

Workflow

Step 1: DNA Extraction

DNA is extracted from clinical samples with commercial extraction kits such as cobas® cfDNA Sample Preparation Kit (not applicable to AML panel), QIAamp® DNA Blood Mini Kit, GeneRead® DNA FFPE Kit(not applicable to AML panel).
40 min (40 min hands-on)

Sanger Sequencing Workflow

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)

Workflow Step 3

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

Quantitative PCR BioRad CFX96

BioRad CFX96


Quantitative PCR ABI 7500

ABI 7500


Quantitative PCR Cobas z480

Cobas z480

Sanger sequencing

Sanger Sequencing ABI 3500 Dx Genetic Analyzer

ABI 3500 Dx Genetic Analyzer

Vartrace AML Panel

Benefits

  • Comprehensive hematological panel: Detects 254 AML hotspot mutations in 6 genes: FLT3, DNMT3A, IDH1, IDH2, KIT, and NPM1
  • Requires 80 ng – 1.5 μg of DNA from whole blood sample
  • Rapid mutation testing: Delivers results in 8 hours, with 2.5 hours of hands-on time

List of Mutations

GeneAA mutationCDS mutation
FLT3p.D835Hc.2503G>C
p.D835Nc.2503G>A
p.D835Yc.2503G>T
p.D835Ac.2504A>C
p.D835Vc.2504A>T
p.D835Ec.2505T>A
p.D835Ec.2505T>G
p.I836Fc.2506A>T
p.I836Lc.2506A>C
p.I836Vc.2506A>G
p.I836Dc.2506_2507delATinsGA
p.I836Hc.2506_2507delATinsCA
p.I836Mc.2508C>G
DNMT3Ap.R882Cc.2644C>T
p.R882Gc.2644C>G
p.R882Sc.2644C>A
p.R882Hc.2645G>A
p.R882Lc.2645G>T
p.R882Pc.2645G>C
IDH1p.R132Cc.394C>T
p.R132Gc.394C>G
p.R132Sc.394C>A
p.R132Hc.395G>A
p.R132Lc.395G>T
p.R132Pc.395G>C
IDH2p.R140Gc.418C>G
p.R140Wc.418C>T
p.R140Lc.419G>T
p.R140Qc.419G>A
p.R172Kc.515G>A
p.R172Mc.515G>T
p.R172Sc.515G>C
KITp.D816Hc.2446G>C
p.D816Yc.2446G>T
p.D816Ic.2446_2447delGAinsAT
p.D816Vc.2447A>T
NPM1p.W288fs*12c.863_864insTCTG
p.W288fs*12c.863_864insCATG
p.W288fs*12c.863_864insCCTG

Sample Data

Product AML Sample Data

Variant verification by Sanger sequencing

Because NuProbe’s BDA technology enriches the variant allele frequency (VAF) of mutations by over 200-fold, our AML sequencing research panel effectively improves Sanger’s limit of detection of 20% VAF to 0.1% VAF. Sanger sequencing can be applied to confirm the identity of quantities detected and quantitated by qPCR using standard sequencing protocols.

Download Product User Manual

    Vartrace BRAF Assay

    Benefits

    • 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

    List of Mutations

    AA mutationCDS mutation
    p.G596Vfs*2c.1787del
    p.G596Dc.1787G>A
    p.L597Sc.1789_1790delinsTC
    p.L597Vc.1789C>G
    p.L597Qc.1790T>A
    p.L597Rc.1790T>G
    p.L597Pc.1790T>C
    p.A598Tc.1792G>A
    p.A598Vc.1793C>T
    p.T599dupc.1794_1796dup
    p.T599delc.1794_1796del
    p.A598_T599insVc.1794_1795insGTT
    p.T599dupc.1795_1797dup
    p.A598_T599insIc.1795_1796insTAA
    p.A598_T599insKKIGDFGLAc.1795_1796insAAAAAATAGGTGATTTTGGTCTAGCTA
    p.T599_V600delinsIKc.1796_1799delinsTAAA
    p.T599Ic.1796C>T
    p.T599Rc.1796C>G
    p.T599_V600insTTc.1797_1798ins?
    p.T599_V600insTTc.1797delinsTACTACG
    p.V600Rc.1797_1799delinsGAG
    p.V600_K601delinsENc.1797_1803delinsTGAGAAT
    p.T599_V600ins2c.1798_1799ins6
    p.V600Lc.1798G>T
    p.V600Mc.1798G>A
    p.V600Kc.1798_1799delinsAA
    p.V600dupc.1798_1800dup
    p.T599_V600insEATc.1798_1799insAGGCTACAG
    p.V600Rc.1798_1799delinsCG
    p.T599_V600insETTc.1798_1799insAGACTACAG
    p.V600Tc.1798_1799inv
    p.V600Rc.1798_1799delinsAG
    p.V600Qc.1798_1799delinsCA
    p.V600Lc.1798G>C
    p.V600Ac.1799T>C
    p.V600Ec.1799_1800delinsAA
    p.V600Ec.1799T>A
    p.V600Dc.1799_1800delinsAT
    p.V600Efs*11c.1799_1800del
    p.V600Gc.1799T>G
    p.V600Dc.1799_1800delinsAC
    p.V600_K601delinsEc.1799_1801del
    p.K601Lc.1801_1802inv
    p.K601Ec.1801A>G
    p.K601delc.1801_1803del
    p.K601Gfs*44c.1801_1816del

    Sample Data

    BRAF mutation testing

    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.

    Download Product User Manual

      Vartrace EGFR Assay

      Benefits

      • Detects 272 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

      List of Detected and Quantitated Mutations

      ExonAA mutationCDS mutation
      18p.G719Sc.2155G>A
      p.G719Cc.2155G>T
      p.G719Ac.2156G>C
      19>20 Exon 19 del mutations
      20p.T790Mc.2369C>T
      p.C797Sc.2389T>A
      p.C797Gc.2389T>G
      p.C797Yc.2390G>A
      p.C797Sc.2390G>C
      p.S768Ic.2303G>T
      >5 Exon 20 insertion mutations
      21p.L858Rc.2573T>G
      p.L861Qc.2582T>A

      Sample Data

      Quantitative PCR
      • 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.
      Quantitative PCR
      • 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.

      Download Product User Manual

        Request Quote / More Info

        Alessandro Pinto, Ph.D., Senior Development Scientist at NuProbe.

        Fill in the form to contact Ale for questions about the VarTrace products.