VX-680 639089-54-6 under the following conditions: 951C for 10 min

under the following conditions: 951C for 10 min, followed by 50 cycles of 941C for 45 s, 601C for 1 min and 721C for 45 s. In all cases, samples were assessed VX-680 639089-54-6 in duplicate. Data were interpreted as follows: if only the control reaction occurred with no mutant reaction, the sample was classified as wild type, if neither reaction occurred, then the sample was classified as unknown, as the concentration of DNA was below the limit of detection, if the mutant reaction occurred, the sample was classified as mutant only if the reaction DCt between control and mutant reaction was smaller than the DCt for each of the control wild type standards on the run to ensure that the mutant reaction was not simply a nonspecific signal.
If there was discordance between the replicates or if the DCt was within 1 Ct of the DCt cutoff, then the experiment for the sample was repeated in triplicate, AB1010 790299-79-5 and the sample was considered positive only if all three replicates were positive. Positive cell line controls were created using DNA extracted from the HT29 cell line, known to be heterozygous for the p.V600E mutation. Human genomic DNA was used as a non mutant DNA containing negative control and appropriate reagent control was used in all PCR runs. All FFPE extracted DNA samples found to be positive for the BRAF mutant by ARMS were sequenced to determine the exact nucleic acid change. Sequencing reactions for tumour DNA Tumour DNA was added to duplicate PCR assays containing primers that amplified BRAF exon 15. The resulting PCR products were sequenced in forward and reverse directions using ABI BigDye sequencing and analysed using SeqScape.
A mutation result was accepted if it was present in both forward and reverse sequencing traces, and in duplicate PCRs. Cloning and sequencing for BRAF mutations To confirm the presence of BRAF mutations in cfDNA from samples in which cfDNA was BRAFt but the matched tumour sample was negative for a mutation by ARMS, cfDNA was extracted from 1ml of serum and cloned and sequenced for the presence of BRAF mutations. Cloning was performed using the TOPO TA Cloning kit with chemically competent Escherichia coli strain TOP 10F,. PCR products containing the BRAF sequence were obtained using the same primer sequences and conditions as those used for exon 15 BRAF sequencing as described above. A mutation result was accepted if it was present in both forward and reverse sequencing traces.
Reproducibility of BRAF detection in cfDNA over 1 year The reproducibility of BRAF detection in cfDNA was tested in 24 serum samples stored at 801C for 6 months, 13 of which were positive for BRAF mutations on initial sampling. A separate set of 24 serum samples stored at 801C for 12 months were re tested for BRAF mutations, 17 of which were positive for BRAF mutations on initial sampling. The reproducibility of BRAF detection in cfDNA stored at 201C for 6 months was tested on 26 samples, 17 of which had tested positive for BRAF mutations at the initial analysis. The reproducibility of BRAF detection in cfDNA stored at 201C for 12 months was tested on a further set of 24 samples, 16 of which had tested positive for BRAF mutations at the initial analysis. Biostatistical analysis The primary end point in study D1532C00003 was PFS and, similar to the primary analysis for this study, a multivariate analysis of PFS was carried out for those patients with serum results, using the Cox proportional hazards model allowing for the effect of treatment and adjustin