The kit consists of 4 PCR assays: one for detecting mutations in codon 719 (exon 18), one for detecting mutations in codons 768 and 790 (exon 20), one for detecting mutations in codons 858 to 861 (exon 21), as well as one for detecting deletions in exon 19. The 4 regions are amplified separately by PCR and sequenced through the defined region (see figure " Illustration of the EGFR assay
"). The amplicon covering codons 768 and 790 is divided into 2 sequencing reactions. Sequences surrounding the defined positions serve as normalization and reference peaks for quantification and quality assessment of the analysis.
After PCR using primers targeting exons 18, 19, 20, and 21, the amplicons are immobilized on Streptavidin Sepharose High Performance beads. Single-stranded DNA is prepared, and the corresponding sequencing primers anneal to the DNA. The samples are then analyzed on the PyroMark Q24 System using a run setup file and a run file. It is recommended to use the EGFR Plug-in Report to analyze the run. This report ensures that the correct LODs are used and different sequences to analyze are automatically used to detect all mutations and deletions. However, the run can also be analyzed using the analysis tool integral to the PyroMark Q24 System (see figures " Pyrogram trace of a normal genotype in codon 719", " Pyrogram trace of a normal genotype in codon 768", " Pyrogram trace of a normal genotype in exon 19", and " Pyrogram trace of a GGAATTAAGAGAAGC deletion in exon 19"). The "Sequence to Analyze" can be then adjusted for detection different deletions in exon 19 and for rare mutations in the other exons after the run.