BRAF Pyro Kit

For sequencing-based detection and quantitation of mutations in the BRAF gene

Features

  • Comprehensive results in real time
  • Accurate quantification of mutations in the BRAF gene
  • Easy detection of complex mutations
  • Sequence context provides built-in control of the assay
  • Flexible post-run analysis
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BRAF Pyro Kit (24)

Cat. No. / ID: 970470

For 24 reactions: Sequencing Primers, PCR Primers, Unmethylated Control DNA, PyroMark PCR Master Mix, CoralLoad Concentrate, Buffers, and Reagents
The BRAF Pyro Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

The BRAF Pyro Kit is a molecular detection kit for the identification of mutations in the BRAF gene. The kit provides primers and reagents for amplification of the BRAF gene, plus buffers, primers, and reagents for detection and quantification of mutations in real time using Pyrosequencing technology on the PyroMark Q24 System.

Principle

The BRAF Pyro Kit is used for quantitative measurements of BRAF mutations in codons 600 and 464–469 of the human BRAF gene in real time using Pyrosequencing technology on the PyroMark Q24 System. The BRAF gene encodes the kinase v-raf murine sarcoma viral oncogene homolog B1, a proto-oncogene that acts downstream of EGFR. Mutations in the BRAF gene are found in 6–8% of all cancers and more frequently in melanoma (50–60%), colorectal cancer (10%), and thyroid (39%) cancers. Approximately 90% of these mutations are V600E substitutions.

Procedure

The product consists of 2 assays: one for detecting mutations in codon 600 and the other for detecting mutations in codons 464–469 (see flowchart "Illustration of the BRAF assay"). The two regions are amplified separately by PCR and sequenced through the defined region. Sequences surrounding the defined positions serve as normalization and reference peaks for quantification and quality assessment of the analysis. 

After PCR using primers targeting codon 600 and codons 464-469, the amplicons are immobilized on Streptavidin Sepharose High Performance beads. Single-stranded DNA is prepared, and the corresponding sequencing primers anneal to the DNA. The samples are then analyzed on the PyroMark Q24 System using a run setup file and a run file (see figures " Pyrogram trace of a normal genotype in codon 600", "Pyrogram trace of a normal genotype in codons 464-469", and "Pyrogram trace of a GTG to GAG mutation in base 2 of codon 600"). The "Sequence to Analyze" can be adjusted for detection of rare mutations after the run.

See figures

Applications

The BRAF Pyro Kit is used for the detection of mutations in codons 600 and 464–469 of the human BRAF gene.

Supporting data and figures

Resources

Analysis Software (1)
BRAF Plug-in 1.3
SOFTWARE (1MB)
Version 1.3.0.7
For use with PyroMark Q24 Software version 2.0.8
Software User Guides (1)
For installation and use with PyroMark Q24 Instruments and PyroMark Q24 Software version 2.0

FAQ

Can I use more than 10ng DNA for the PCR reaction for the Pyro kits?
The NRAS Pyro, EGFR Pyro. BRAF PyroKRAS Pyro, and UGT1A1 Pyro Kits all require that no more than 10ng of DNA be used for the PCR reaction.  Too much DNA can sometimes lead to inhibition problems in the PCR reaction.
FAQ ID -2379
Which purification kits are recommended for the Pyro kits?

The purification kits recommended for the BRAF Pyro, EGFR Pyro, KRAS Pyro, and NRAS Pyro kits are:

Paraffin-embedded tissue

Blood

FAQ ID -2380
What plug-ins are available for the Pyrosequencing kits?
Plug-Ins are available for:
  • EGFR Pyro Kit 
  • KRAS Pyro Kit
  • NRAS Pyro Kit
  • RAS extension Kit
  • BRAF Pyro Kit
  • These plug-ins are available for download on the respective catalog page under the Product Resources tab in the Analysis Software section. Note: PyroMark Q24 software version 2.0.7 is needed for the usage of the Plug-ins.
    FAQ ID -2381
    Is V600E the only mutation that can be detected with the codon 600 assay in the BRAF Pyro Kit?
    No, the V600E is not the only mutation that can be detected with the BRAF Pyro Kit.  The sequence to analyze can be changed post-run to allow detection also of V600M (GTG>ATG), V600A (GTG>GCG) and V600G (GTG>GGG).
    FAQ ID -2382
    Can PyroMark Gold reagents be vortexed?
    Reconstiuted enzyme and substrate of PyroMark Gold Reagents, should not be vortexed since this could lead to conformational changes which affect the activity.
    FAQ ID -2844