Application Guide - QIAxcel Advanced

QIAxcel Advanced Application Guide 10/2016 41 Real-time PCR Amplification was performed using the therascreen® EGFR Pyro Kit* on a real-time PCR system. As per the manufacturer’s protocol, a 25 µl PCR mix was prepared containing 30 ng of template DNA and 8 µM of primers. The initial denaturation step was at 95°C for 15 min, followed by 42 amplification cycles of denaturation at 95°C for 20 s, annealing at 53°C for 30 s, and elongation at 72°C for 20 s. The final elongation step was at 72°C for 10 min. Electrophoresis and DNA size estimation After amplification, the PCR products were separated using the QIAxcel Advanced, the QIAxcel DNA High Resolution Kit, and the 0M800 method. Amplicon size determination was done with the QX Alignment Marker 15 bp/600 bp and QX DNA Size Marker 25–500 bp. Data were analyzed and visualized using the QIAxcel ScreenGel® software. Pyrosequencing was then performed. Results PCR fragments were amplified from EGFR exon 19 and c-kit exon 11 using samples with known deletions compared to the wild type (Figure 1 and 2). Analyses using the QIAxcel Advanced System showed high accuracy in identifying wild-type and mutated DNA fragments based on the size estimation. Human EGFR exon 19 has an amplicon size of about 250 bp. When there were deletions, extra bands could be seen in the gel images (Figure 1). Human c-kit exon 11 has an amplicon size of about 220 bp. Mutations were found at various sizes (Figure 2). All of the samples with deletion mutations were detected and the corresponding deletion size was correctly scored, allowing for the exclusion of wild-type samples from subsequent Pyrosequencing. Figure 1. Detection of deletion mutations in EGFR exon 19. Human EGFR exon 19 has an amplicon size of 250 bp. Lanes A1 and A3 are from samples with EGFR exon 19 deletions and have extra bands, while the remaining lanes are from wild-type EGFR. A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 – 600 – 500 – 400 – 300 – 250 – 200 – 75 – 50 – 25 – 15 – 150 – 100 [bp/nt]

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