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BRAF Pyro Kit

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

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.

Cat No./ID: 970470
BRAF Pyro Kit (24)
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For 24 reactions: Sequencing Primers, PCR Primers, Unmethylated Control DNA, PyroMark PCR Master Mix, CoralLoad Concentrate, Buffers, and Reagents
0
Pyrogram trace of a normal genotype in codon 600.
"Sequence to Analyze" CWCTGTAGC.
1
Pyrogram trace of a normal genotype in codons 464-469.
"Sequence to Analyze" CTGTTNCAAATGATHCAGATHCA.
2
Pyrogram trace of a GTG to GAG mutation in base 2 of codon 600.
Nucleotide 1799, indicated with an arrow. "Sequence to Analyze" CWCTGTAGC.
3
Illustration of the BRAF Pyro Kit assay.
The sequence indicated is the analyzed sequence for a normal sample. FP and FPB: Forward PCR primers (B indicates biotinylation); RP and RPB: Reverse PCR primers (B indicates biotinylation); Seq: Sequencing primers.
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.

Applications

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

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