42 of the Chromas software package (Conor McCarthy, Southport, Australia). For all analyses, data obtained PLX3397 purchase with the forward and reverse primers were combined and aligned to the consensus sequence obtained from the BLAST GenBank database http://www.ncbi.nlm.nih.gov/nuccore/166706780?report=genbank. Figure 1 Sequencing of the KRAS gene in DNA isolated from NSCLC tissues. (A) Wild type-(12Gly-GGT, 13Gly-GGC), (B) Mutant- (12Asp-GAT). Pyrosequencing In the pyrosequencing method for DNA sequence analysis [16, 17], inorganic phosphate released in the course of nucleotide incorporation serves as the initial substrate in a sequence of four
successive enzymatic reactions. This result in the emission of light, which functions as a signal that is proportional to the number of nucleotides incorporated. In this project, the PyroMark K-ras assay test (Biotage, Uppsala, Sweden) was used for primary amplification P005091 cost and pyrosequencing of both the 12th and the 13th codons of the KRAS oncogene (Figure 2). The following amplification program was used: the mixture was heated at 95°C for 5 min, then subjected to 45 cycles of 95°C for
15 s, 57°C for 30 s, and 72°C for 15 s. It was then held at 72°C for 5 min, and finally cooled to and held at 4°C. The final concentrations of the PCR components were: 1x PCR buffer, 2 mM MgCl2, 0.125 mM dNTPs, 0.2 μM FW primer and 0.2 μM REV biotinylated primer, 1U of AmpliTaq polymerase (Perkin Elmer, Waltham, USA) and 2 ng/μl DNA template. Fifteen μl of the PCR product was run on a 1,5% agarose gel (Sigma-Aldrich, St. Louis, USA) to confirm successful amplification, and 100 ng of PCR products were sent to the EpigenDX company (Worcester, USA) to be analyzed using the PyroMark MD System and the Pyromark ID analysis Software with previously validated cut-off of
5%. Figure 2 Pyrosequencing of the KRAS gene in DNA isolated from NSCLC tissues. (A) Wild type-(12Gly-GGT, 13Gly-GGC), (B) Mutant-KRAS (12Cys-TGT). K-RAS TheraScreen DxS The TheraScreen DxS KRAS Mutation Kits KR-21 and KR-22 (QiaGen, Hilden, Germany) are designed to detect six mutations in codon 12 (Gly > Ala, Asp, Arg, Cys, Ser, and Val) and one in codon selleck screening library 13 (Gly > Asp) of the KRAS oncogene. The primers used in the assay have two characteristic features: I-BET-762 research buy sequence-specific 3’ ends (which comprise the PCR-Amplification Refractory Mutation System, PCR-ARMS®) to identify specific mutations, and Real-time PCR-Scorpion® primer tags, which fluoresce when incorporated into double-stranded DNA (Figure 3). The commercial test kit includes an internal reaction control and a synthetic control template. The degree of mutation of KRAS is calculated on the basis of the difference between the control reaction and the allele-specific reaction in terms of the number of cycles required for the fluorescence of the reaction mixture to exceed the background level (Δ-CT) . Figure 3 TheraScreen analysis of the KRAS gene in DNA isolated from NSCLC tissue. (A) Wild type.