Qiagen, 970590, RAS Extension Pyro Kit (24)

For 24 reactions: Sequencing Primers, PCR Primers, Unmethylated Control DNA, PyroMark PCR MasterMix, CoralLoad Concentrate, and Buffers and Reagents

Features

- Cover mutations in KRAS Codons 59, 61, 117 and 146
- Cover mutations in NRAS Codons 12, 13, 59, 61, 117 and 146
- Software plug-in for interpretation of complex sequence information
- Reliable results in short turnaround time

Product Details

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.

Performance

LOD of the mutations covered by the RAS Extension Pyro Kit are ranging from 1.8% to 8.8%.

Principle

Codon: Nucleic acid substitution
KRAS codon 59 (GCA): 175G>A
176C>G: A59G
KRAS codon 61 (CAA): 183A>C
182A>T: Q61L
182A>G: Q61R
183A>T: Q61H
181C>G: Q61E
KRAS codon 117 (AAA): 351A>C
351A>T: K117N
KRAS codon 146 (GCA): 436G>A
436G>C: A146P
437C>T: A146V
NRAS codon 12 (GGT): 34G>A
34G>T: G12C
34G>C: G12R
35G>A: G12D
35G>T: G12V
35G>C: G12A
NRAS codon 13 (GGT): 37G>A
37G>T: G13C
37G>C: G13R
38G>A: G13D
38G>T: G13V
38G>C: G13A
NRAS codon 59 (GGT): 175G>A
176C>G: A59G
NRAS codon 61 (CAA): 181C>A
182A>G: Q61R
182A>T: Q61L
183A>T: Q61H
183A>C: Q61H
183A>G: Q61Q
NRAS codon 117 (AAG): 351G>C
351G>T: K117N
NRAS codon 146 (GCC): 436G>A
436G>C: A146P
437C>T: A146V

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:

Procedure

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.

Applications

The RAS Extension Pyro Kit enables quantitative mutation analysis applicable to several fields of research, including cancer research and epigenetics. The kit is for the quantitative detection of mutations in codons 59, 61, 117, and 146 of the human KRAS oncogene and codons 12, 13, 59, 61, 117, and 146 of the human NRAS oncogene, using DNA extracted from formalin-fixed paraffin-embedded (FFPE) human tissue.