Qiagen, 870801, BRAF RGQ PCR Kit (24)

For 24 reactions: Control Assay, 4 Mutation Assays, Positive Control, Taq DNA Polymerase, Water for NTC, and Water for Sample Dilution

Features

- Robust test that can detect low levels of mutant DNA
- Ready-to-use system with simple workflow
- Selective and sensitive detection using ARMS and Scorpions technologies

Principle

- V600E (GAG) and V600E complex (GAA)
- V600D (GAT)
- V600K (AAG)
- V600R (AGG)

The 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 5plex HRM instrument.

The BRAF RGQ PCR Kit enables detection of the following mutations against a background of wild-type genomic DNA.

The kit detects the presence of the V600E (GAG) and V600E complex (GAA) but does not distinguish between them.

The BRAF RGQ PCR 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 BRAF RGQ PCR Kit comprises 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 to detect the presence or absence of mutated DNA.

Applications

The BRAF RGQ PCR Kit enables detection of the following mutations against a background of wild-type genomic DNA.