Massarray maldi tof system

For PGx analyses, venous blood samples were obtained using EDTA containing Vacutainers. DNA extraction and PGx analyses were performed by Labor Risch molecular genetics laboratory, Bern-Liebefeld, Switzerland. DNA was extracted using the QIAsymphony DSP DNA Mini Kit according to the manufacturer’s instructions. The SNPs of CYP2C19 of the isolated DNA were subsequently amplified by means of the iPLEX assay which consists of multiplex-PCR, SAP reaction, and iPLEX primer extension. The modified products were then separated using the MassARRAY MALDI-TOF System by Agena Bioscience. The analysis included the following CYP2C19 associated SNPs: rs12248560 (*17), rs28399504 (*4), rs41291556 (*8), rs4244285 (*2), rs4986893 (*3), rs56337013 (*5), rs72552267 (*6), and rs72558186 (*7).
Analyzed genotypes were subsequently forwarded to SONOGEN and further processed by its XP expert system. SONOGEN XP provides an automated report with classification of the genotype into a corresponding metabolizer phenotype based on established star allele nomenclature, as well as predictions of interactions between the identified gene variants and current or potential future pharmacotherapy.

From each tumour sample 6 × 10 µM sections were cut from the paraffin block. Hematoxylin and eosin staining was performed on the first and last sections and checked by an experienced Pathologist to ensure that there was tumour present. If the tumour content was lower than 30%, tumour area was macrodissected. DNA was extracted using an All Prep DNA FFPE kit (Qiagen), as per manufacturer’s instructions. DNA concentration was calculated using the Qubit dsDNA kit.
Mass Spectrometry single nucleotide polymorphism genotyping technology (Agena Biosciences, Hamburg, Germany) was applied to DNA extracted from the FFPE samples to detect a total of 108 nonsynonymous somatic mutations in PIK3CA, EGFR, ERBB2, ERBB3 and ERBB4. Mutations in ERBB-family genes were identified using publically available data from the TCGA database and a literature search [23 (link)]. AVSIFT and Mutation Assessor scores were used to determine the ERBB-family mutations that were likely to be deleterious, and result in activation of the PI3K and MAPK signalling pathways. The full list of mutations is listed in the supplementary data (Additional File 1: Table S1). Matrix chips were analysed on an Agena MassArray MALDI-TOF system. Visual inspection and Typer Software were used to identify genotypes based on mass spectra. Reactions where > 15% of the resultant mutant mass ran in the mutant site were scored as positive.

Based on the FST analysis results of this study and referring to previous results (26 (link)), primer design was performed using the upstream and downstream 200 bp sequence information of nsSNPs of common genes significantly selected by XP-CLR in the top 5% of FSTs. The primers were designed using the Agena online software Design Suite 2.0.¹ The designed primer sequence was derived and then synthesized by Invitrogen. The mutation sites were identified by stroma-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The genotyping system used was the MassARRAY® MALDI-TOF system developed by Agena.