Gender, age, and tumor location distribution of cases used in <i>KRAS</i> and <i>BRAF</i> mutation analysis.

<p>Gender, age, and tumor location distribution of cases used in <i>KRAS</i> and <i>BRAF</i> mutation analysis.</p

Allele-specific forward primers allow detection of single and double <i>BRAF</i> mutations.

<p>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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198795#pone.0198795.s001" target="_blank">S1 Table</a>) were diluted and used as PCR templates.</p

Detection of BRAF V600E mutations in reference standards by AS-PCR assays.

<p>Detection of BRAF V600E mutations in reference standards by AS-PCR assays.</p

DNA sample quality and mutation status of study samples.

<p>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 <i>BRAF</i> mutation in poor-quality DNA obtained from FFPE CRC tissue (Sample 5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198795#pone.0198795.t003" target="_blank">Table 3</a>). Genomic DNA sample from HEK-293 cells were used as negative control. D. <i>KRAS</i> and <i>BRAF</i> mutation status of 178 FFPE CRC tissues. The samples analyzed by methods other than cobas® KRAS Mutation Test, AmoyDx® <i>KRAS</i> Mutation Detection Kit and our AS-PCR assay were excluded from this study. mut^- = mutation not detected, mut⁺ = mutation detected, ND = not determined.</p

Identification of <i>BRAF</i> mutations in ultra-short DNA.

<p>A and B. Detection of BRAF V600E1 mutation (A) and V600K mutation (B) in 45-, 35-, or 25-base synthesized oligonucleotides (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198795#pone.0198795.s001" target="_blank">S1 Table</a>), 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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198795#pone.0198795.s001" target="_blank">S1 Table</a>). Allele-specific primers for BRAF V600E and V600K were combined in the same quantitative PCR reaction.</p

Development of ultra-short PCR assay to reveal <i>BRAF</i> V600 mutation status in Thai colorectal cancer tissues

<div><p>The protein kinase BRAF is one of the key players in regulating cellular responses to extracellular signals. Somatic mutations of the <i>BRAF</i> gene, causing constitutive activation of BRAF, have been found in various types of human cancers such as malignant melanoma, and colorectal cancer. BRAF V600E and V600K, most commonly observed mutations in these cancers, may predict response to targeted therapies. Many techniques suffer from a lack of diagnostic sensitivity in mutation analysis in clinical samples with a low cancer cell percentage or poor-quality fragmented DNA. Here we present allele-specific real-time PCR assay for amplifying 35- to 45-base target sequences in <i>BRAF</i> gene. Forward primer designed for BRAF V600E detection is capable of recognizing both types of BRAF V600E mutation, i.e. V600E1 (c.1799T>A) and V600E2 (c.1799_1800delTGinsAA), as well as complex tandem mutation caused by nucleotide changes in codons 600 and 601. We utilized this assay to analyze Thai formalin-fixed paraffin-embedded tissues. Forty-eight percent of 178 Thai colorectal cancer tissues has <i>KRAS</i> mutation detected by highly sensitive commercial assays. Although these DNA samples contain low overall yield of amplifiable DNA, our newly-developed assay successfully revealed BRAF V600 mutations in 6 of 93 formalin-fixed paraffin-embedded colorectal cancer tissues which <i>KRAS</i> mutation was not detected. Ultra-short PCR assay with forward mutation-specific primers is potentially useful to detect BRAF V600 mutations in highly fragmented DNA specimens from cancer patients.</p></div

Characteristics of <i>BRAF</i> V600E mutation-positive cases in Thai colorectal cancer tissues.

<p>Characteristics of <i>BRAF</i> V600E mutation-positive cases in Thai colorectal cancer tissues.</p