The KRAS RGQ PCR Kit is a ready-to-use kit for the detection of 7 somatic mutations in the KRAS oncogene using real-time PCR on the Rotor-Gene Q 5plex HRM instrument. 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.
The KRAS RGQ PCR Kit utilizes two technologies — ARMS (Amplification Refractory Mutation System) and Scorpions — for detection of mutations in real-time PCR.
Allele- or mutation-specific amplification is achieved using ARMS. Taq DNA polymerase (Taq) 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.
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. During PCR when the probe binds to the amplicon, the fluorophore and quencher become separated. This leads to an increase in fluorescence from the reaction tube.