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KRAS Pyro Kit

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

Features

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

Cat. No. / ID: 970460

For 24 reactions: Sequencing Primers, PCR Primers, Unmethylated Control DNA, PyroMark PCR Master Mix, CoralLoad Concentrate, Buffers, and Reagents.
The KRAS 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

Product Details

The KRAS Pyro Kit is a molecular detection kit for the identification of mutations in the KRAS gene. The kit provides primers and reagents for amplification of the KRAS 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 KRAS Pyro Kit is used for quantitative measurements of mutations in codons 12, 13, and 61 of the human KRAS gene in real time using Pyrosequencing technology on the PyroMark Q24 System. The KRAS gene encodes a protein that plays a critical role in the EGFR signaling cascade. Mutations in the KRAS gene can affect how the protein stimulates these downstream pathways. KRAS is mutated in approximately 30% of all cancer types. Cancers that exhibit a high frequency of KRAS mutation include colorectal cancer (35%) and lung (18%) cancer.

The following mutations are detected:

  • Codon 12/13: G12A, G12C, G12D, G12R, G12S, G12V, G13D
  • Codon 61: Q61E, Q61H, Q61L, 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 KRAS assay" and " Pyrogram trace of a normal genotype in codons 12 and 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 KRAS Plug-in Report should be used to analyze the run. This report ensures that the correct LODs are used and different sequences to analyze are automatically used to detect all mutations. However, the run can also be analyzed using the analysis tool integral to the PyroMark Q24 System (see figure " Pyrogram trace of a GGT to GAT mutation in base 2 of codon 12"). The "Sequence to Analyze" can be then adjusted for detection of rare mutations after the run (see figures " Pyrogram trace of a GGT to TGT mutation in base 1 of codon 12" and " Pyrogram trace of the reanalysis of the sample in the previous figure").

See figures

Applications

The KRAS Pyro Kit enables accurate, quantitative mutation analysis applicable to several fields of research, including cancer research and epigenetics. The kit is used for detecting the most common mutations in codons 12, 13, and 61 of the human KRAS gene.

The following mutations are detected:

  • Codon 12/13: G12A, G12C, G12D, G12R, G12S, G12V, G13D
  • Codon 61: Q61E, Q61H, Q61L, Q61R

Supporting data and figures

Resources

Analysis Software (1)
KRAS Plug-in 1.3
SOFTWARE (1MB)
Version 1.3.0.6
For use with PyroMark Q24 Software version 2.0.8
Safety Data Sheets (1)
Download Safety Data Sheets for QIAGEN product components.
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
How do I analyze codon 61 mutations with the RAS extension kit (cat. no. 971590)?

Mutations in KRAS codon 61 must be analyzed separately with the KRAS Plug-in and not with the RAS extension plug-in.

FAQ ID - 3622
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