therascreen KRAS RGQ PCR Kit

For the detection of 7 somatic mutations in the KRAS oncogene

Features

Compliance with EU IVD Directive 98/79/EC
Sensitive, robust test that can detect low levels of mutant DNA
Ready-to-use system with simple workflow
Highly selective and sensitive due to ARMS and Scorpions PCR technologies

Product Details

The therascreen KRAS RGQ PCR Kit is a molecular diagnostic kit for detection of 7 somatic mutations in the KRAS gene using real-time PCR on the Rotor-Gene Q 5plex HRM instrument. The kit provides all necessary reagents optimized for rapid and sensitive detection of a low percentage of mutant DNA in a background of wild-type genomic DNA. ARMS PCR technology combined with the Scorpions detection technology provides exceptionally high sensitivity and specificity for the detection of specific mutations.

Principle

The therascreen KRAS RGQ PCR Kit is intended to detect 7 mutations in codons 12 and 13 of the KRAS gene. 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.

KRAS mutations detected by the therascreen KRAS RGQ PCR Kit include:

12ALA

12ASP

12ARG

12CYS

12SER

12VAL

13 ASP

The kit utilizes two technologies — ARMS (Amplification Refractory Mutation System) and Scorpions — for detection of mutations in real-time PCR.

ARMS

Allele- or mutation-specific amplification is achieved by ARMS. Taq DNA polymerase is effective at distinguishing between a match and a mismatch at the 3' end of a PCR primer. Specific mutated sequences are selectively amplified, even in samples where the majority of the sequences do not carry the mutation. When the primer is fully matched, the amplification proceeds with full efficiency. When the 3' base is mismatched, only low-level background amplification occurs.

Scorpions

Detection of amplification is performed using Scorpions. Scorpions are bifunctional molecules containing a PCR primer covalently linked to a probe. The fluorophore in this probe interacts with a quencher, also incorporated into the probe, that reduces fluorescence. When the probe binds to the amplicon during PCR, the fluorophore and quencher become separated. This leads to an increase in fluorescence from the reaction tube.

Procedure

The therascreen KRAS RGQ PCR Kit uses a two-step procedure. The first step is performance of the control assay to assess the total DNA in a sample. The second step is to complete the mutation assay for the presence or absence of mutated DNA.

Applications

The therascreen KRAS RGQ PCR Kit is intended to detect 7 mutations in codons 12 and 13 of the KRAS gene. Studies have shown that KRAS mutation testing can better define which metastatic colorectal cancer patients will benefit from treatment with epidermal growth factor receptor (EGFR) inhibiting monoclonal antibodies, such as panitumumab and cetuximab.