Application Guide - QIAxcel Advanced

42 QIAxcel Advanced Application Guide 10/2016 Figure 2. Deletion and duplication mutations in c-kit exon 11. Human c-kit exon 11 has an amplicon size of about 220 bp. Lane A11: sample with an exon 11 homozygous deletion. Lanes A2, A7, and A10: the wild-type c-kit exon 11 allele. Lane A12: sample with an exon 9 duplication (p.A502 Y503dup). [bp/nt] A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 – 600 – 500 – 400 – 300 – 250 – 200 – 150 – 100 – 75 – 50 – 25 – 15 Conclusions • The QIAxcel system proved to be highly suitable for screening amplicons prior to Pyrosequencing using EGFR and c-kit as the model systems. All of the deletion mutants were detected and the corresponding deletion size was correctly scored, allowing the exclusion of wild-type samples from the downstream sequencing step. • The described QIAxcel-based screening method yields robust and reproducible results of sufficiently high quality for more efficient downstream Sanger sequencing and Pyrosequencing applications. • This method using the QIAxcel system can be applied to increase the speed and reduce the costs of deletion/insertion studies. References 1. Willmore, C., Holden, J.A., Zhou, L., Tripp, S., Wittwer, C.T., and Layfield, L.J. (2004) Detection of c-kit–activating mutations in gastrointestinal stromal tumors by high-resolution amplicon melting analysis. Am. J. Clin. Pathol. 122, 206. 2. Hirota, S. and Isosaki, K. (2006) Pathology of gastrointestinal stromal tumors. Pathol. Int. 56, 1. 3. Lopes, L.F. and Bacchi, C.E. (2007) EGFR and gastrointestinal stromal tumor: an immunohistochemical and FISH study of 82 cases. Mod. Pathol. 20, 990.

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