Figure 1
Figure 1. RT-PCR assay for the detection of BRAFV600E. (A) BRAFV600E real-time quantification assay. (i) Assays for BRAFwt and BRAFmut were almost identical, with the exception of the BRAFV600E mutation-specific primer, which includes the nucleotide substitution of the T > A at the 3′end and an additional mismatch nucleotide (A > G at the third position from the 3′end) that further enhances the specific detection of mutated transcripts. Forward and reverse primer are underlined. Mutated nucleotide and mismatch nucleotide in the forward primer are given in red. Green represents fluorescein-labeled detection probe; and red, LC640-labeled probe. In detail, the reaction was performed in a final volume of 20 μL by use of 2 μL mastermix (LightCycler Fast Start DNA Master Hybridization Probes; Roche Diagnostics), 4mM MgCl2, 0.5μM of each forward (BRAFwt-F: TAGGTGATTTTGGTCTAGCTACAGT or BRAFmut-F: TAGGTGATTTTGGTCTAGCTACGGA) and reverse primer (BRAF-R: TCTGACTGAAAGCTGTATGGATT; Metabion), 0.25μM of each of the 2 fluorescent hybridization probes (BRAF-Fl: GTGGGTCCCATCAGTTTGAACAG-fluorescein and BRAF-LCred640: LC640-GTCTGGATCCATTTTGTGGATGGCACC-phosphate), and 2 μL cDNA (accounting for an equivalent of mRNA of 200 000 cells). Amplification was performed after initial incubation at 95°C for 10 minutes in a 3-step cycle procedure (denaturation 95°C, 1 second, ramp rate 20°C/s, annealing temperature 64°C, 10 seconds, ramp rate 20°C/s, and extension 72°C, 26 seconds, ramp rate 2°C/s) for 45 cycles. (ii) Position of primers (BRAF-F and BRAF-R), V600E mutation, and hybridization probes at the mRNA are indicated. (iii) For plasmid standards, exons 14 to 16 of BRAFwt and BRAFmut each were cloned into a pCR2.1TOPO vector. (B) Ten-fold dilution series of the plasmid carrying the BRAFV600E: 200 000, 2 plasmid copies. (C) Standard curve showing efficiency of the BRAFV600E specific PCR of 1.93. (D) Ten-fold dilution of a diagnostic BRAFV600E mutated patient sample in cDNA of an unmutated patient showing a sensitivity of 1 of 100 000. BRAFwt signal is indicated as “nonspecific background.”

RT-PCR assay for the detection of BRAFV600E. (A) BRAFV600E real-time quantification assay. (i) Assays for BRAFwt and BRAFmut were almost identical, with the exception of the BRAFV600E mutation-specific primer, which includes the nucleotide substitution of the T > A at the 3′end and an additional mismatch nucleotide (A > G at the third position from the 3′end) that further enhances the specific detection of mutated transcripts. Forward and reverse primer are underlined. Mutated nucleotide and mismatch nucleotide in the forward primer are given in red. Green represents fluorescein-labeled detection probe; and red, LC640-labeled probe. In detail, the reaction was performed in a final volume of 20 μL by use of 2 μL mastermix (LightCycler Fast Start DNA Master Hybridization Probes; Roche Diagnostics), 4mM MgCl2, 0.5μM of each forward (BRAFwt-F: TAGGTGATTTTGGTCTAGCTACAGT or BRAFmut-F: TAGGTGATTTTGGTCTAGCTACGGA) and reverse primer (BRAF-R: TCTGACTGAAAGCTGTATGGATT; Metabion), 0.25μM of each of the 2 fluorescent hybridization probes (BRAF-Fl: GTGGGTCCCATCAGTTTGAACAG-fluorescein and BRAF-LCred640: LC640-GTCTGGATCCATTTTGTGGATGGCACC-phosphate), and 2 μL cDNA (accounting for an equivalent of mRNA of 200 000 cells). Amplification was performed after initial incubation at 95°C for 10 minutes in a 3-step cycle procedure (denaturation 95°C, 1 second, ramp rate 20°C/s, annealing temperature 64°C, 10 seconds, ramp rate 20°C/s, and extension 72°C, 26 seconds, ramp rate 2°C/s) for 45 cycles. (ii) Position of primers (BRAF-F and BRAF-R), V600E mutation, and hybridization probes at the mRNA are indicated. (iii) For plasmid standards, exons 14 to 16 of BRAFwt and BRAFmut each were cloned into a pCR2.1TOPO vector. (B) Ten-fold dilution series of the plasmid carrying the BRAFV600E: 200 000, 2 plasmid copies. (C) Standard curve showing efficiency of the BRAFV600E specific PCR of 1.93. (D) Ten-fold dilution of a diagnostic BRAFV600E mutated patient sample in cDNA of an unmutated patient showing a sensitivity of 1 of 100 000. BRAFwt signal is indicated as “nonspecific background.”

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