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KRAS Pyro Kit

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

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.

Cat No./ID: 970460
KRAS Pyro Kit (24)
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.
0
Pyrogram trace of a normal genotype in codons 12 and 13.
1
Pyrogram trace of a GGT to GAT mutation in base 2 of codon 12.
Nucleotide 35, indicated with an arrow.
2
Pyrogram trace of a GGT to TGT mutation in base 1 of codon 12.

The Pyrogram shows the mutation (nucleotide 34 indicated with an arrow) with the "Sequence to Analyze" GNTGRCGTAGGC targeting base 2 in codon 12 (nucleotide 35). A yellow color indicates that this sequence is unexpected and needs to be checked.

3
Pyrogram trace of the reanalysis of the sample in the previous figure.
The mutation GGT to TGT was reanalyzed with the "Sequence to Analyze" NGTGRCGTAGGC targeting base 1 in codon 12 (nucleotide 34).
4
Illustration of the KRAS 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 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").

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

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Kit Handbooks (2)
For quantitative measurements of mutations in codons 12, 13, and 61 of the human KRAS gene
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Software User Guides (1)
For installation and use with PyroMark Q24 Instruments and PyroMark Q24 Software version 2.0.7
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Protocol Files (1)
Version 1.2.1.5
For use with PyroMark Q24 Software version 2.0.7
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Safety Data Sheets (1)
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