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NRAS Pyro Kit (24)
Cat. No. / ID: 970530
For 24 reactions: Sequencing Primers, PCR Primers, Unmethylated Control DNA, PyroMark PCR Master Mix, CoralLoad Concentrate, Buffers, and Reagents
The NRAS Pyro Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
✓ 24/7 automatic processing of online orders
✓ Knowledgeable and professional Product & Technical Support
✓ Fast and reliable (re)-ordering
Features
- Comprehensive results in real time
- Accurate quantification of mutations in the NRAS gene
- Easy detection of complex mutations
- Sequence context provides built-in control of the assay
- Flexible post-run analysis
Product Details
The NRAS Pyro Kit is a molecular detection kit for the identification of mutations in the NRAS gene. The kit provides primers and reagents for amplification of the NRAS 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 NRAS Pyro Kit is used for quantitative measurements of mutations in codons 12, 13, and 61 of the human NRAS gene in real time using Pyrosequencing technology on the PyroMark Q24 System. The NRAS gene encodes a GTPase neuroblastoma RAS viral (v-ras) oncogene homolog. Mutations in NRAS are found in approximately 13–25% of all malignant melanomas, occurring frequently in codons 12, 13, and 61. These mutations results in the constitutive activation of NRAS signaling pathways.
The following mutations are detected:
- Codon 12/13: G12A, G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13D, G13R, G13S, G13V
- Codon 61: Q61E, Q61H, Q61K, Q61L, Q61P, Q61R
Procedure
The product consists of 2 assays: one for detecting mutations in codons 12 and 13 and the second for detecting mutations in codon 61 (see figures " Illustration of the NRAS assay" and " Pyrogram trace of a normal genotype in codon 12–13"). 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 codons 12/13 and codon 61, 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. The "Sequence to Analyze" can be then adjusted for detection of rare mutations after the run (see figures " Pyrogram trace of a GGT to AGT mutation in base 1 of codon 12" and " Pyrogram trace of the reanalysis of the sample in the previous figure").
See figures
Applications
The NRAS Pyro Kit is used for the detection of mutations in codons 12, 13, and 61 of the human NRAS gene. The kit enables accurate, quantitative mutation analysis applicable to several fields of research, including cancer research and epigenetics.>
The following mutations are detected:
- Codon 12: G12A, G12C, G12D, G12R, G12S, G12V
- Codon 13: G13A, G13C, G13D, G13R, G13S, G13V
- Codon 61: Q61E, Q61H, Q61K, Q61L, Q61P, Q61R
Supporting data and figures
Pyrogram trace of a normal genotype in codon 12-13.
Pyrogram trace of a normal genotype in codon 12-13.
Resources
Kit Handbooks (7)
Safety Data Sheets (1)
Analysis Software (1)
Certificates of Analysis (1)
FAQ
Can I use more than 10ng DNA for the PCR reaction for the Pyro kits?
Which purification kits are recommended for the Pyro kits?
What plug-ins are available for the Pyrosequencing kits?
Can PyroMark Gold reagents be vortexed?