therascreen BRAF Pyro Kit

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

Features

  • Compliance with EU IVD Directive 98/79/EC
  • Comprehensive results in real time
  • Accurate quantitation of cancer-related BRAF gene mutations
  • Easy interpretation of complex sequence information
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therascreen BRAF Pyro Kit (24)

Cat. No. / ID: 971470

For 24 reactions: Sequencing Primers, PCR Primers, Unmethylated Control DNA, PyroMark PCR Master Mix, CoralLoad Concentrate, and therascreen Buffers and Reagents
€1,778.00
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The therascreen BRAF Pyro Kit is intended for in vitro diagnostic use.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

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Product Details

The therascreen BRAF Pyro Kit is a molecular detection kit for the identification of mutations in the BRAF gene. The kit contains 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.

Performance

Linearity

Linearity was determined using mixtures of plasmids carrying the wild type or mutant sequence for the mutation V600E (GTG->GAG) in codon 600 of the BRAF gene (see figure " Linearity of V600E mutation"). The plasmids were mixed in proportions to give four levels of mutation (5, 10, 30, and 50%). Each mixture was analyzed with three different lots of the therascreen BRAF Pyro Kit in three Pyrosequencing runs with three replicates each.

The results were linear within an allowable nonlinearity of 5 % units in the tested range of 5 to 50% mutation level.

Precision

The precision data allows the determination of the total variability of the assays and was obtained at three different levels by analysis of the above mentioned plasmid mixtures with three replicates each.

Repeatability (intra-assay and inter-batch variability) was calculated based on the data for determination of linearity (three runs on the same day using varying lots of the therascreen BRAF Pyro Kit). Intermediate precision (intra-laboratory variability) was determined in three runs within one laboratory on three different days with varying operators, PyroMark Q24 systems, and lots of the therascreen BRAF Pyro Kit. Reproducibility (inter-laboratory variability) was calculated from two runs each in an internal and external laboratory and using varying lots of the therascreen BRAF Pyro Kit.

Precision estimates are expressed as standard deviation of the measured mutation frequencies in % units. The repeatability, intermediate precision, and reproducibility for the mutation V600E (GTG->GAG) in codon 600 was 0.6–2.1, 0.7–1.8, and 0.8–2.1 % units, respectively, in the measured range of 5 to 50% mutation level.

Precision values for the mutation V600E (GTG->GAG) in codon 600

% mutated plasmid

Repeatability (Mean, SD)

Intermediate precision (Mean, SD)

Reproducibility (Mean, SD)

5

5.2, 0.6 4.4, 0.7 5.1, 0.8
10

9.1, 1.0

9.6, 1.0 9.6, 1.3
30 28.1, 2.1 27.9, 1.8 28.3, 2.1
50 46.9, 1.2 46.3, 1.5 47.9, 1.7
All values are given as % units. SD: standard deviation (n=9). % mutated plasmid based on OD260 measurement.
See figures

Principle

The therascreen BRAF Pyro Kit is used for quantitative measurements of 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.

The following mutations are detected:

  • Codon 600: V600A, V600E, V600G, and V600M
  • Codon 464–469: G464E, G464V, G466E, G466V, G469A, G469E, and G469V

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 figure " 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 therascreen BRAF Pyro Kit is intended to be used as an aide to identify melanoma or colorectal cancer patients more likely to benefit from treatment with cancer therapies that are selective inhibitors of BRAF. The kit is used for the detection of mutations in codons 600 and 464–469 of the human BRAF gene.

The following mutations are detected:

  • Codon 600: V600A, V600E, V600G, and V600M
  • Codon 464–469: G464E, G464V, G466E, G466V, G469A, G469E, and G469V

Supporting data and figures

Resources

Safety Data Sheets (1)
Download Safety Data Sheets for QIAGEN product components.
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 my therascreen Pyro Kits?
It is not recommended to use more than 10ng for the PCR reaction in the therascreen BRAF, therascreen EGFR, therascreen KRAStherascreen NRAS Pyro, and therascreen UGT1A1 Kits.  Too much DNA can sometimes lead to inhibition problems in the PCR reaction.
FAQ ID -2385
Is V600E the only mutation that can be detected with the therascreen BRAF Pyro Kit codon 600 assay?
No, the V600E is not the only mutation that can be detected with the therascreen BRAF Pyro Kit codon 600 assay.  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 -2388
Which purification kits are recommended for the therascreen Pyro kits?

 

Which purification kits are recommended for the therascreen Pyro kits?

 

FAQ ID -2386
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
What plug-ins are available for the therascreen Pyro kits?

Plug-Ins are available for:


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 -2387