EGFR RGQ PCR Kit Version 2

The EGFR RGQ PCR Kit is a research use only assay for detection of specific mutations, insertions, and deletions in the EGFR 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 high sensitivity and specificity for the detection of specific mutations.

The EGFR RGQ PCR Kit enables detection of 29 somatic mutations in the EGFR gene. The EGFR gene encodes the epidermal growth factor receptor (EGFR) protein. Mutations in the tyrosine kinase domain of the EGFR gene can enable tumor growth and progression. EGFR mutations are found in approximately 10% of non-small cell lung cancer incidences in the US and 35% in East Asia. Additionally, EGFR mutations are found in 6% of brain tumors.

The EGFR RGQ PCR Kit enables detection of 29 somatic mutations in the EGFR gene including:

• 19 deletions in exon 19
• T790M 
• L858R
• L861Q
• G719X
• S768I

• 3 insertions in exon 20

The kit utilizes two technologies — ARMS  (Amplification Refractory Mutation System) and Scorpions — for detection of EGFR mutations using real-time PCR on the Rotor-Gene Q 5plex HRM instrument.

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.

The EGFR 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.

The EGFR RGQ PCR Kit enables detection of 29 somatic mutations in the EGFR gene including:

• T790M, G719X, S768I
• L858R, L861Q
• 3 insertions in exon 20
• 19 deletions in exon 19