NRAS Pyro Kit

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

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
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.

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

Resources

Analysis Software (1)
RAS Extension Plug-in 1.3
SOFTWARE (1MB)
Version 1.3.0.7
For use with PyroMark Q24 Software version 2.0.8

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
    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