The therascreen BRAF RGQ PCR Kit constitutes a ready-to-use kit for the detection of somatic mutations in the BRAF oncogene using polymerase chain reaction (PCR) on the Rotor-Gene Q MDx 5plex HRM instrument. The BRAF gene encodes the kinase V-raf murine sarcoma viral oncogene homolog B1, a proto-oncogene that acts downstream of EGFR. The kit enables qualitative measurement of somatic mutations in the BRAF oncogene from genomic DNA extracted from formalin-fixed paraffin-embedded (FFPE) samples.
The therascreen BRAF RGQ PCR Kit enables detection of the following mutations against a background of wild-type genomic DNA.
- V600E (GAG) and V600E complex (GAA)
- V600D (GAT)
- V600K (AAG)
- V600R (AGG)
The kit detects the presence of the V600E (GAG) and V600E complex (GAA) but does not distinguish between them.
The therascreen BRAF 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 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.
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.