Description
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.
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
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").
