Fig 1.
Allele-specific forward primers allow detection of single and double BRAF mutations.
A. Schematic diagrams of primer design. Forward primers were chosen to specifically amplify BRAF V600E1 (c.1799T>A), V600E2 (c.1799_1800delTGinsAA) and V600K (c.1798_1799delGTinsAA) mutations. Detection of BRAF V600K mutation can be performed separately or together with BRAF V600E mutation detection. WT = wild-type. B and C. Detection of low abundant BRAF V600E1 (B) and V600K mutations (C) in diluted DNA reference standards, which allelic frequencies were measured by droplet digital PCR. DNA amplification is monitored at each cycle of PCR using SYBR Green reagent included in PCR premix. D and E. Detection of BRAF V600E2 mutation (D) and complex tandem mutation caused by nucleotide changes in codons 600 and 601 (BRAF V600E1 and K601E mutations) (E). Synthesized oligonucleotides (S1 Table) were diluted and used as PCR templates.
Table 1.
Detection of BRAF V600E mutations in reference standards by AS-PCR assays.
Fig 2.
Identification of BRAF mutations in ultra-short DNA.
A and B. Detection of BRAF V600E1 mutation (A) and V600K mutation (B) in 45-, 35-, or 25-base synthesized oligonucleotides (S1 Table), which mimic severely fragmented single-stranded DNA. C. Cross-reactivity with non-target mutations, i.e. BRAF V600A, V600D, V600G, V600M, and V600R, presented in 45-base oligonucleotide templates (S1 Table). Allele-specific primers for BRAF V600E and V600K were combined in the same quantitative PCR reaction.
Fig 3.
DNA sample quality and mutation status of study samples.
A and B. Assessment of 200-bp (A) and 41-bp (B) amplifiable DNA in study samples. DNA samples isolated from cell line (control), FFPE reference standards and FFPE CRC tissues were evaluated. We included the control samples in every run to allow comparisons between runs. The amount of amplifiable DNA was compared to control by comparing the cycle thresholds (Ct) obtained from the quantitative PCR results. Error bars indicate the standard error of the mean. * = statistically significant t-test result (p< 0.01), NS = not statistically significant. Ref Std = FFPE reference standards, CRC = colorectal cancer tissue specimens, Ct = cycle threshold. C. Detection of BRAF mutation in poor-quality DNA obtained from FFPE CRC tissue (Sample 5 in Table 3). Genomic DNA sample from HEK-293 cells were used as negative control. D. KRAS and BRAF mutation status of 178 FFPE CRC tissues. The samples analyzed by methods other than cobas® KRAS Mutation Test, AmoyDx® KRAS Mutation Detection Kit and our AS-PCR assay were excluded from this study. mut- = mutation not detected, mut+ = mutation detected, ND = not determined.
Table 2.
Gender, age, and tumor location distribution of cases used in KRAS and BRAF mutation analysis.
Table 3.
Characteristics of BRAF V600E mutation-positive cases in Thai colorectal cancer tissues.