Wt human genomic dna

LOD of the SensiScreen^® FFPE BRAF qPCR Assay was evaluated by serial dilutions of cell line DNA, harboring BRAF mutations, in a background of 50 ng WT human genomic DNA (Promega, Madison, Wisconsin, USA). Five different concentrations of mutated DNA were tested (10%, 5%, 2%, 1% and 0.5%) on MyGo Pro real-time PCR instruments (IT-IS Life Science Ltd, Mahon, Ireland) and Rotor-Gene 6000 (Corbett Research), using the SensiScreen^® protocol previously described [22 (link)]. The SensiScreen^® FFPE BRAF qPCR Assay limit of blank (LOB) was evaluated by application of 20 replica of 50 ng WT human genomic DNA (Promega, Madison, Wisconsin, USA).

DNA oligonucleotides were purchased from Sigma-Aldrich (Ishikari, Japan), and stored as 100 mM stock solutions in 10 mM Tris-HCl with 1 mM EDTA (pH 8.0). WT human genomic DNA was purchased from Promega (Tokyo, Japan). Heterozygote human genomic DNA with seven mutations (G12A, G12C, G12D, G12R, G12S, G12V and G13D) in the KRAS gene codon 12 and 13 were obtained from Horizon Diagnostics (Cambridge, UK). Eprobes (shown in Table I) were obtained from K.K. DNAFORM (Yokohama, Japan). Point mutations in codon 12 of the KRAS gene (GAT, GCT, GTT, AGT, TGT) were cloned into the plasmid pGEM-T (Promega) as previously described (26 (link)) and stored in glycerol at −80°C for long-term conservation. Point mutation of codon 13 (GAC) in the KRAS gene was prepared using the same protocol. The KRAS codon 12 (CGT) point mutation was prepared using the synthetic DNA ordered and inserted into a pUC57 plasmid (GenScript, Tokyo, Japan). The sequences of WT and all seven mutant clones were verified on ABI PRISM 3100 Avant (Applied Biosystems, Tokyo, Japan), diluted in TE buffer and stored at −20°C until use.