Description
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.
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
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").
