Streptavidin sepharose high performance beads

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), were used for RAS mutations testing, according to the manufacturer’s instructions. Briefly, 5 μl of template DNA (2–10 ng of genomic DNA) were 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 were 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). The sequences were analyzed using Pyromark Q24 software in the AQ analysis mode. In each run, a 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.

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.