Description
The RAS Extension Pyro Kit is a molecular detection kit for identification of mutations in the exons 3 and 4 of the KRAS oncogene and exons 2, 3 and 4 of the human NRAS oncogene. The kit contains primers and reagents for amplification of the KRAS and NRAS gene plus buffers, primers, and reagents for detection and quantification of mutations in real time using Pyrosequencing technology on the PyroMark Q24 System.
LOD of the mutations covered by the RAS Extension Pyro Kit are ranging from 1.8% to 8.8%.
The RAS Extension Pyro Kit is used for quantitative measurements of mutations in exons 3 and 4 of the human KRAS oncogene, and exons 2, 3, and 4 of the human NRAS oncogene using Pyrosequencing technology on the PyroMark Q24 System. The RAS genes encode proteins that play a critical role in the EGFR signaling cascade. Mutations in the RAS genes can affect how the proteins stimulate these downstream pathways. The following mutations are detected: Codon Nucleic acid substitution Amino acid substitution KRAS codon 59 (GCA) 175G>A A59T 176C>G A59G KRAS codon 61 (CAA) 183A>C Q61H 182A>T Q61L 182A>G Q61R 183A>T Q61H 181C>G Q61E KRAS codon 117 (AAA) 351A>C K117N 351A>T K117N KRAS codon 146 (GCA) 436G>A A146T 436G>C A146P 437C>T A146V NRAS codon 12 (GGT) 34G>A G12S 34G>T G12C 34G>C G12R 35G>A G12D 35G>T G12V 35G>C G12A NRAS codon 13 (GGT) 37G>A G13S 37G>T G13C 37G>C G13R 38G>A G13D 38G>T G13V 38G>C G13A NRAS codon 59 (GGT) 175G>A A59T 176C>G A59G NRAS codon 61 (CAA) 181C>A Q61K 182A>G Q61R 182A>T Q61L 183A>T Q61H 183A>C Q61H 183A>G Q61Q NRAS codon 117 (AAG) 351G>C K117N 351G>T K117N NRAS codon 146 (GCC) 436G>A A146T 436G>C A146P 437C>T A146V
For a general overview of Pyrosequencing technology, please visit our Pyrosequencing resource center The RAS Extension Pyro Kit consists of 8 assays covering the different regions of interest (see Figure 1. Assays of the RAS Extension Pyro Kit and Figure 2. Pyrogram trace obtained after analysis of a sample with a wild-type genotype with the KRAS 146 assay.). The 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 the different codons, 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 RAS Extension Plug-in Report should be used to analyze the run. This report ensures that the correct limit of detection (LOD) values are used and different sequences to analyze are automatically used to detect all mutations (see Figure 3. RAS Extension Plug-In Report menu and Figure 4. RAS Extension Plug-In Report). However, the run can also be analyzed using the analysis tool integral to the PyroMark Q24 System. The "Sequence to Analyze "can be then adjusted for detection of rare mutations after the run.
