Targeted Sequencing of RAS Mutations

The analysis of RAS mutations was performed using the TheraScreen® KRAS Pyro Kit (for KRAS codons 12 and 13) and the TheraScreen® RAS Extension Pyro Kit (for KRAS codons 59/61, 117, and 146 and NRAS codons 12, 13, 59, 61, 117, and 146) (Qiagen, Germany), according to the manufacturer's instructions. As described previously [2 (link)], 5 µl of template DNA (2–10 ng of genomic DNA) was amplified by polymerase chain reaction (PCR) in a 20 µl volume containing 12.5 µl of PyroMark® PCR Master Mix 2x, 2.5 µl of Coral Load Concentrate 10x, 4 µl of nuclease-free water, and 1 µl of the corresponding set of PCR primers (Qiagen). 10 µl of PCR products was immobilized to Streptavidin Sepharose High-Performance beads (Qiagen) to prepare the single-stranded DNA. The corresponding sequencing primers were allowed to anneal to the DNA using a PyroMark Q24 plate and a vacuum workstation (Qiagen). PyroMark Q24 reagents (enzyme mixture, substrate mixture, and nucleotide all from Qiagen) were prepared and loaded into a cartridge to be dispensed during the sequencing process. Finally, the sequences were analyzed using PyroMark Q24 software in the AQ analysis mode. In each run, two controls were included: negative control (without template DNA) and an unmethylated control DNA, provided by the kit as a positive control for PCR, and sequencing reactions were included.