Concentrations of DNA samples CX-6258 ic50 were measured spectrophotometrically using a NanoDrop ND 1000 spectrophotometer (NanoDropTechnologies, Wilmington, USA). Genotyping methods Analyses were performed according to a blinded design, in which the experimentalist was not aware
of the KRAS mutation status of any given sample. 131 NSCLC samples were analyzed using four methods: Direct sequencing, Pyrosequencing, and the TheraScreen DxS and K-ras StripAssay kits. Due to limited amount of tissue, only 116 samples from this group were also subjected to HRM analysis and 114 yielded usable data. Significance of the concordance of mutation detection with different methods for two categories (wildtype and mutant) was assessed by κ statistics ( http://faculty.vassar.edu/lowry/kappa.html). Direct sequencing method Two primers were used to prepare amplicons for use in Sanger
dideoxy termination sequencing : a forward (FW) primer, 5′AAA AGG TAC TGG TGG AGT ATT TGA, and selleck chemical a 3’ reverse (REV) primer, 5′ TCA TGA AAA TGG TCA GAG AAA CC 3′ (Generi-Biotech, Hradec Králové, Czech Republic). PCR was performed with a reaction volume of 50 μl in an MJ Research PTC-200 find more Peltier Thermal Cycler (Watertown, USA). The composition of the PCR reaction mixture was as follows: MgCl2 (3 mM, ThermoScientific, Waltham, USA), dNTPs (0.2 mM, ThermoScientific), ThermoStart DNA polymerase ADP ribosylation factor (2U, ThermoScientific), FW-primer (0.3 μM), REV-primer (0.3 μM), 1xPCR buffer, and between 10 ng and 100 ng of genomic DNA per reaction. The following amplification program was used: 95°C/15 min to activate the Taq polymerase; 35x (95°C/30 s, 58°C/30 s 72°C/30 s) for denaturation, annealing, and extension; and finally 75°C/5 min to finalize the extension, followed by cooling to 15°C. The PCR product was separated using a 2% agarose gel and purified using the QIAquick PCR purification kit (QIAGEN, Hilden, Germany). For each sample specimen, separate sequencing reactions were performed
using the forward (FW) and reverse (REV) primers. The sequencing primers were internal to the amplicons from the previous PCR cycles: FW – 5′ TTA ACC TTA TGT GTG ACA TGT TCT AA 3′, REV – 5′ AGA ATG GTC CTG CAC CAG TAAT 3′. Sequencing reactions were performed according to the manufacturer’s protocol in a 20 μl reaction volume containing 4 μl DTCS Quick Start kit (Beckman Coulter, Brea, USA), 1 μl (10 μM) of the FW or REV primer, 10 μl nuclease-free water, and 5 μl of 25x diluted template PCR product. After cleaning, precipitated DNA was diluted in SLS-formamide (Beckman Coulter, Brea, USA) and dideoxylabelled fragments were size-separated using an automated CEQ 8800 Genetic Analysis System (Beckman Coulter, Brea,USA) (Figure 1).