The therascreen NRAS Pyro Kit is a molecular detection kit for identification of mutations and deletions in the NRAS gene. The kit contains 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.
Linearity Linearity was determined using mixtures of plasmids carrying the wild-type or mutant sequence for the mutations GGT->GAT in codons 12 and 13 and the mutation CAA->CGA in codon 61 (see figure "Linearity of GGT->GAT mutation"). The plasmids were mixed in proportions to give four levels of mutation (5, 10, 30, and 50%). Each mixture was analyzed with three different lots of the therascreen NRAS Pyro Kit in three Pyrosequencing runs with three replicates each. The results were linear within an allowable nonlinearity of 5 % units in the tested range of 5 to 50% mutation level. Similar results were obtained for the mutations GGT->GAT in codon 13 and CAA->CGA in codon 61. Precision The precision data allows the determination of the total variability of the assays and was obtained at three different levels by analysis of the above mentioned plasmid mixtures with three replicates each. Repeatability (intra-assay and inter-batch variability) was calculated based on the data for determination of linearity (three runs on the same day using varying lots of the therascreen NRAS Pyro Kit). Intermediate precision (intra laboratory variability) was determined in three runs within one laboratory on three different days with varying operators, PyroMark Q24 instruments, and lots of the therascreen NRAS Pyro Kit. Reproducibility (inter-laboratory variability) was calculated from two runs each in an internal and external laboratory and using varying lots of the therascreen NRAS Pyro Kit. The repeatability, intermediate precision, and reproducibility for the mutation GGT>GAT in codon 12 was 1.2–1.9, 1.0–2.0, and 1.3–3.1 % units, respectively, in the measured range of 5–50% mutation level. Similar results were obtained for the mutations GGT>GAT in codon 13 and CAA>CGA in codon 61. Precision for the mutation GGT>GAT in codon 12 % mutated plasmid Repeatability (Mean, SD) Intermediate precision (Mean, SD) Reproducibility (Mean, SD) 5 7.5, 1.2 7.3, 1.0 6.7, 1.3 10 14.6, 1.3 13.5, 1.1 13.7, 1.3 30 37.8, 1.9 37.9, 1.5 36.1, 2.9 50 59.8, 1.7 60.4, 2.0 57.5, 3.1 All values are given as % units. % mutated plasmid based on OD 260 measurement, SD: standard deviation (n=9 for repeatability and intermediate precision, n=12 for reproducibility).
The therascreen 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 after reanalysis of the sample in the previous figure").
