75 units AmpliTaqGOLD (ABI), 200 μM dNTP (ABgene) and supplemented with bovine serum albumin (New England VEGFR inhibitor Biolabs) with 5′ end tagged primers (forward primer tag: ACTGTAAAACGACGGCCAGT; reverse primer tag: selleck screening library ACCAGGAAACAGCTATGACC) that amplified BRAF exon 15, and NRAS exon 2: BRAF exon 15 forward TTTCCTTTACTTACTACACCTC, reverse CTTTCTAGTAACTCAGCAGCATC; NRAS exon 2 forward CCCCCAGGATTCTTACAGAA; reverse ATACACAGAGGAAGCCTTCG. PCRs were conducted using the following cycling conditions: 95°C, 10 min, (94°C, 30 s, 58°C, 30 s, 72°C, 1 min) × 40 cycles, 72°C, 10 min. EGFR analysis was conducted on NSCLC DNA samples. Five microlitres of tumour DNA diluted 1/5 in water was added to triplicate

PCR assays containing PCR buffer II at 2 mM MgCl2, 3.75 units

AmpliTaqGOLD (ABI), 200 μM dNTP (ABgene) and supplemented with bovine serum albumin (New England Biolabs) with 5′ end tagged primers (forward primer tag: ACTGTAAAACGACGGCCAGT; reverse primer tag: ACCAGGAAACAGCTATGACC) that amplified EGFR exons 18 to 21: EGFR exon 18 forward CCTTCCAAATGAGCTGGCAAGTG, reverse TCTCACAGGACCACTGATTACTG; EGFR exon 19 forward GCAGCATGTGGCACCATCTCAC, reverse find more CAGGGTCTAGAGCAGAGCAGC; EGFR exon 20 forward CGCATTCATGCGTCTTCACCTG, reverse CTATCCCAGGAGCGCAGACCG; EGFR exon 21 forward TCGACGTGGAGAGGCTCAGAG and reverse CTGCGAGCTCACCCAGAATGTC. PCRs were conducted using the flowing conditions: 95°C 10 min, (94°C, 20 s, 61°C, 30 s (dropping 0.5°C/cycle), 72°C, 1 min) × 13 cycles, (94°C, 20 s, 57°C, 30 s, 72°C,1 min) × 30 cycles, 72°C, 10 min. Resulting PCR products were bidirectionally sequenced using primers complimentary to the Forward and Reverse tags

on the primary PCR primers using ABI Big Dye sequencing, and analysed using Mutation Surveyor software (SoftGenetics). To eliminate eltoprazine false positive mutations occurring due to sample fixation artefacts, a mutation result was only accepted if it was present in at least two out of three independent PCRs in at least one of each Forward and Reverse sequencing traces. Results Melanoma analysis Out of the 177 melanoma samples extracted, 163 (92%) were successfully analysed by ARMS as indicated by the presence of the control reaction, and 156 (88%) were successfully analysed by DNA sequencing as indicated by readable sequencing traces. In total, 69 BRAF mutations were detected using a combination of both methods; 67 of these were at codon 600, one at codon 601 (K601E) and another at codon 581 (N581S). The 67 codon 600 mutations (1799T>A) were detected using the ARMS assay but only 46 of these were detected by DNA sequencing. Forty-one of these were V600E mutations and five were V600K. The BRAF 1799T>A ARMS assay could detect V600E, V600K and V600D mutations as they all contain mutations at the same nucleotide position, but could not distinguish between them.