Journal: Diagnostics

Article Title: Analytical Evaluation of an NGS Testing Method for Routine Molecular Diagnostics on Melanoma Formalin-Fixed, Paraffin-Embedded Tumor-Derived DNA

doi: 10.3390/diagnostics9030117

Figure Lengend Snippet: Control DNA samples obtained from cell line mixes in different proportions.

Article Snippet: Finally, wild type samples were analyzed by castPCR (competitive allele-specific TaqMan PCR; Thermo Fisher Scientific, Inc., Waltham, MA, USA) technology by using the BRAF_476_mu (Assay ID: Hs00000111_mu) probes with the corresponding wild type allele assays BRAF_476_wt (Assay ID: Hs00000110_wt) and the gene reference assay BRAF_rf (Assay ID: Hs00000172_rf) in a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific).

Techniques: Control, Variant Assay

Journal: Diagnostics

Article Title: Analytical Evaluation of an NGS Testing Method for Routine Molecular Diagnostics on Melanoma Formalin-Fixed, Paraffin-Embedded Tumor-Derived DNA

doi: 10.3390/diagnostics9030117

Figure Lengend Snippet: Results of the analysis performed by Ion Reporter for the expected variants in cell MIX C at the lowest VAF tested.

Article Snippet: Finally, wild type samples were analyzed by castPCR (competitive allele-specific TaqMan PCR; Thermo Fisher Scientific, Inc., Waltham, MA, USA) technology by using the BRAF_476_mu (Assay ID: Hs00000111_mu) probes with the corresponding wild type allele assays BRAF_476_wt (Assay ID: Hs00000110_wt) and the gene reference assay BRAF_rf (Assay ID: Hs00000172_rf) in a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific).

Techniques: Variant Assay

Journal: bioRxiv

Article Title: Unveiling the Domain-Specific and RAS Isoform-Specific Details of BRAF Regulation

doi: 10.1101/2023.04.24.538112

Figure Lengend Snippet: HRAS and KD D594G disrupt BRAF autoinhibition. (A) Western blot of pulldown assay of pre-incubated His/MBP-NT1 and His-KD added to purified active GST-HRAS on glutathione resin. Representative data of 2 independent replicates with similar results. (B) OpenSPR experiments in which NT1 at 5, 15, 44, 133, 400 nM (black) and NT1 + HRAS-GMPPNP (1:1) at 5, 15, 44, 133, and 400 nM (red) flowed over KD immobilized on carboxyl sensors. Representative data of 3 independent replicates with similar results. (C) OpenSPR experiments of NT1 at 5, 15, 44, 133, and 400 nM flowed over immobilized KD WT (black) or KD MUT (D594G; red) on carboxyl sensors. Representative data of 3 independent replicates with similar results. (D) Western blot of purified His/MBP-NT1 binding to either biotinylated His-KD WT or His-KD MUT (D594G) in pulldown assays on streptavidin beads. Representative data of 3 independent replicates with similar results.

Article Snippet: BRAF-KD-WT/D594G was immobilized (50 ug/ml; ~6000 RU) on a carboxyl sensor chip (Nicoya) following standard manufacturer protocols of the amine coupling kit (Nicoya).

Techniques: Western Blot, Incubation, Purification, Binding Assay

Journal: bioRxiv

Article Title: Unveiling the Domain-Specific and RAS Isoform-Specific Details of BRAF Regulation

doi: 10.1101/2023.04.24.538112

Figure Lengend Snippet: Model of BRAF activation. (A) BRAF is initially an autoinhibited monomer in the cytosol, in which signaling through the RAS-RAF-MEK-ERK cascade is not promoted. The BRAF N-terminal region (NT1; aa 1–288), including the BRAF Specific Region (BSR), Cysteine Rich Domain (CRD), and RAS Binding Domain (RBD), interacts with active GTP-bound RAS at the membrane in an isoform specific manner. BRAF has higher affinity for KRAS due to isoform differences, as shown through the different dissociation constants (K D ) determined through OpenSPR. (B) Once bound to active H- or K-RAS, BRAF is unable to remain in the autoinhibited conformation and is subsequently activated upon dimerization, which stimulates signaling for events such as cell growth, proliferation, and differentiation. (C) Tight binding is observed with the BRAF Kinase Domain (KD; aa 442–723) and BRAF NT1, revealing the concerted action of the BSR, RBD, and CRD domains to reinforce the autoinhibited conformation and restrict signaling without upstream activation. (D) Oncogenic BRAF D594G stimulates activation of MAPK pathway through a decreased ability to remain in the autoinhibited conformation and an increased potential to dimerize with CRAF. Evading autoinhibitory regulation leads to overactivation of the signaling cascade and tumorigenesis.

Article Snippet: BRAF-KD-WT/D594G was immobilized (50 ug/ml; ~6000 RU) on a carboxyl sensor chip (Nicoya) following standard manufacturer protocols of the amine coupling kit (Nicoya).

Techniques: Activation Assay, Binding Assay, Membrane

Journal: Nature medicine

Article Title: BRAF V600E -induced senescence drives Langerhans cell histiocytosis pathophysiology

doi: 10.1038/s41591-021-01304-x

Figure Lengend Snippet: a, Cartoon shows the experimental breeding scheme used to generate BRAF-V600EScl+ mice and control littermates. d1–5 indicates day 1–5. b, Percentage of RosaYFP+ cells among purified hematopoietic progenitors in BRAF-WTScl+ mice 4 weeks after tamoxifen injections (n = 5 mice). CMP, common myeloid progenitor; MEP, megakaryocytic erythroid progenitor. c, Representative computerized tomography image of a femur from a BRAF-V600EScl+ animal. The red arrow shows a pathognomonic granuloma. d, H&E staining and CD207 immunohistochemistry staining of tissues isolated from BRAF-V600EScl+ mice. We note the presence of multinucleated giant cells and granuloma-like lesions. The graph represents the number of CD207+ cells per mm2 in tissue sections isolated from animals (n = 3 mice per group). e, Percentage of the CD11c+MHCII+ MNP population among RosaYFP+ cells in organs of BRAF-V600EScl+ mice and control littermates. Data are representative of three experiments (n = 7 mice). f, Absolute numbers of total CD45+ immune cells and CD11c+MHCII+ MNPs that populate the lung and the skin of BRAF-V600EScl+ mice and control littermates. Data are representative of three experiments (n = 3–7 mice). NS, not significant. g, Cartoon shows the scheme used to generate the humanized LCH mouse. h, Liver, lung and spleen tissue sections isolated from humanized mice reconstituted with BRAF-V600Ehu HPCs were stained with anti-CD207 and anti-CD1a antibodies. Graphs represent the number of CD207+ or CD1a+ cells per mm2 in tissue sections isolated from animals (n = 3–4 mice). Data are represented as mean ± s.e.m.; statistical significance was analyzed by unpaired two-sided t-tests and is indicated by *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. These data are representative of three independent experiments.

Article Snippet: BRAF V600E or BRAF WT chimeras were generated by transplantation of 1–3 million whole BM cells flushed from the femurs of 8-week-old BRAF -V600E Scl + or BRAF -WT Scl + mice following tamoxifen injections into lethally irradiated (650 cGy × 2) CD45.1 mice aged 8–12 weeks (C57BL/6 background, Charles River Laboratories).

Techniques: Control, Purification, Tomography, Staining, Immunohistochemistry, Isolation

Journal: Nature medicine

Article Title: BRAF V600E -induced senescence drives Langerhans cell histiocytosis pathophysiology

doi: 10.1038/s41591-021-01304-x

Figure Lengend Snippet: a-f, BRAFV600EScl+ mice and BRAFwtScl+ mice were generated as described in Fig. 1a. a, Representative images of spleen, lung, and femurs at 4 weeks post tamoxifen injections. b, Liver and spleen weights. c, Absolute number of BM cells in BRAFV600EScl+ mice and control littermates (n = 10 mice per group). d, Hematoxylin and eosin staining and CD207 immunohistochemistry staining of tissues isolated from BRAFwtScl+ mice (n = 2–3 mice per group). e, Absolute numbers of lung, dermal and epidermal immune cells in BRAFV600EScl+ mice and control littermates. Data are representative of 3 experiments (n = 3–8). f, Percentage of immune cell populations among lung and skin infiltrating RosaYFP+ cells in BRAFV600EScl+ mice and control littermates. Data are representative of 3 experiments (n = 3–8). g, Scheme of the lentiviral vector constructs used to transduce human CD34+ HPC. h, Graph shows that the transduction efficiency of BRAFV600E and NGFR lentiviral constructs in human CD34+ HPC does not exceed 40 %. i, Western blot of BRAF and phospho-ERK performed on purified BRAFV600Ehu and NGFRhu human CD34+ HPC 7 days after transduction. j, Representative flow plot showing the percentage of CD11c+ CD14+ MNP among circulating blood cells in NSG mice reconstituted with BRAFV600Ehu and NGFRhu HPC. Graph represents the percentage of CD11c and or CD14+ cells among human CD45+ cells from NSG circulating blood (n = 2–3 mice per group). Data are represented as mean ± s.e.m; statistical significance analyzed by an unpaired two-sided t-test is indicated by *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: BRAF V600E or BRAF WT chimeras were generated by transplantation of 1–3 million whole BM cells flushed from the femurs of 8-week-old BRAF -V600E Scl + or BRAF -WT Scl + mice following tamoxifen injections into lethally irradiated (650 cGy × 2) CD45.1 mice aged 8–12 weeks (C57BL/6 background, Charles River Laboratories).

Techniques: Generated, Control, Staining, Immunohistochemistry, Isolation, Plasmid Preparation, Construct, Transduction, Western Blot, Purification

Journal: Nature medicine

Article Title: BRAF V600E -induced senescence drives Langerhans cell histiocytosis pathophysiology

doi: 10.1038/s41591-021-01304-x

Figure Lengend Snippet: a–c, BRAF-V600EScl+ mice were generated as described in Fig. 1a, and, in these mice, RosaYFP+ cells mark BRAF-V600E+ cells, while RosaYFP− cells mark BRAF-V600E− cells. In BRAF-WTScl+ control littermates, RosaYFP+ cells mark those that underwent a Cre recombination as well. a, Graphs show the percentage of HSCs, MPPs, common myeloid progenitors, granulocytic myeloid progenitors and megakaryocytic erythroid progenitors among RosaYFP+ cells in the BM of BRAF-V600EScl+ mice and BRAF-WTScl+ control littermates (n = 6–7 mice). b, Percentages of neutrophils, Ly6Chi monocytes (mono), macrophages and dendritic cells among RosaYFP+ cells in the BM of BRAF-V600EScl+ mice and BRAF-WTScl+ control littermates (n = 4–6 mice). c, CFU assays were performed on purified lineage-negative BM cells isolated from BRAF-V600EScl+ mice or BRAF-WTScl+ control littermates (n = 3 mice) cultured in the presence of methylcellulose, with representative phase contrast photomicrographs of colonies. GEMM, granulocytes, erythrocytes, megakaryocytes, monocytes, megakaryocytes. d–f, Purified CD34+ HPCs transduced with BRAFV600E (BRAF-V600Ehu) or NGFR control (NGFRhu) lentiviral vectors were cultured in stem cell medium for 8 d. d, Graphs show the percentages of GMPs (defined as CD34+CD38+CD10−CD123hi/intCD45RA+/−)34 and the percentages of MNPs (defined as CD66b−CD11c+MHCII+) among live cells (n = 3 donors). e, CFU assays in methylcellulose performed on purified BRAF-V600E−GFP− and BRAF-V600E+GFP+ cells isolated from HPCs transduced with the BRAFV600E lentiviral vector or purified NGFR+GFP+ or NGFR−GFP− cells isolated from HPCs transduced with the NGFR lentiviral vector as described in Extended Data Fig. 3 (n = 6 donors). Representative phase contrast photomicrographs of colonies are shown. f, Human CD34+ HPCs transduced with BRAFV600E or the control NGFR lentiviral vector were cultured for 7 d in stem cell medium and analyzed using microarray sequencing. Heatmap representation of genes involved in granulopoiesis and the macrophage–dendritic cell (DC) lineage is shown (n = 4 independent donors per group). g, Purified CD34+ BM cells were isolated from a healthy pediatric donor (in duplicate) and from three patients with LCH and BM mononuclear cells known to be BRAF-V600E+, and these cells were analyzed using bulk RNA-seq. Heatmap representation of genes involved in granulopoiesis and the macrophage–dendritic cell lineage is shown. h,i, BRAF-V600EScl+ mice and BRAF-WTScl+ control littermates were generated as described in Fig. 1a. The percentages of GMPs (h), neutrophils, Ly6Chi monocytes, macrophages and dendritic cells (i) among BRAFWT;RosaYFP− cells in the BM of BRAF-V600EScl+ mice and BRAF-WTScl+ control littermates is shown. j, Human CD34+ HPCs were transduced with BRAFV600E or NGFR lentiviral constructs and cultured in stem cell medium. Seven days later, the percentages of CD11c+ and/or CD14+ MNP cells among BRAFV600E (GFP+) or BRAFWT (GFP−) cells were measured by flow cytometry. Because the transduction efficiency was around 40% for both vectors, GFP+ cells mark BRAF-V600E+ or NGFR+ cells, while GFP− cells mark BRAFWT or NGFR− cells among HPCs transduced with the BRAFV600E or NGFR lentiviral construct, respectively (n = 8 independent donors). k, Healthy human CD34+ cord blood HPCs were cultured in stem cell medium (concentration is represented by the white triangle) in addition to supernatant isolated from HPCs transduced with the BRAFV600E lentiviral construct or the NGFR control (concentration is represented by the black triangle). The graph shows the percentage of HPC differentiation into CD14+ MNPs analyzed by flow cytometry after 5 d of culture (n = 4 independent donors), analyzed by paired t-tests. Data are represented as mean ± s.e.m.; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (unpaired two-sided t-tests, two-way ANOVA test for d and paired two-sided t-tests for j,k). Data are representative of at least three independent experiments.

Article Snippet: BRAF V600E or BRAF WT chimeras were generated by transplantation of 1–3 million whole BM cells flushed from the femurs of 8-week-old BRAF -V600E Scl + or BRAF -WT Scl + mice following tamoxifen injections into lethally irradiated (650 cGy × 2) CD45.1 mice aged 8–12 weeks (C57BL/6 background, Charles River Laboratories).

Techniques: Generated, Control, Purification, Isolation, Cell Culture, Transduction, Plasmid Preparation, Microarray, Sequencing, RNA Sequencing, Construct, Flow Cytometry, Concentration Assay

Journal: Nature medicine

Article Title: BRAF V600E -induced senescence drives Langerhans cell histiocytosis pathophysiology

doi: 10.1038/s41591-021-01304-x

Figure Lengend Snippet: a, Lineage-negative BM cells isolated from either BRAF-V600EScl+CD45.2+ or BRAF-WTScl+CD45.2+ mice were injected intravenously together with lineage-negative CD45.1+ BM cells into lethally irradiated CD45.1+ mice at a 2:1 ratio (CD45.2+:CD45.1+). Graph shows the ratio of CD45.2+:CD45.1+ cells circulating in the blood in each group, measured 4 weeks after transplantation using flow cytometry (n = 8 animals). b–e, BM cells were isolated from BRAF-V600EScl+ and BRAF-WTScl+ control littermates and analyzed for the percentage of BrdU expression in lineage-negative Sca-1+c-Kit+ cells (LSK), megakaryocytic erythroid progenitors, common myeloid progenitors and GMPs after seven daily BrdU injections (1 mg per day) (n = 3–4 mice) (b); Cdkn2a expression from purified lineage-negative BM cells measured by quantitative PCR with reverse transcription (RT–qPCR) (normalized to actin expression) (n = 8 mice) (c); and SAβGal activity of purified lineage-negative BM cells (d). The graph shows the percentage of SAβGal+ cells among total cells (n = 3 mice per group). e, IL-1α, IL-1β and IL-6 protein levels in the BM supernatant, detected by ELISA, are shown (n = 4–6 mice per group). f–i, Liver tissue sections were obtained from BRAF-V600EScl+ and BRAF-WTScl+ control littermates and analyzed for Ki-67+ (violet) cells among liver infiltrating CD207+ LCH cells (red) (f). Graphs show the percentage of Ki-67+ cells among CD207+ cells in liver sections (n = 3 mice per group), Cdkn2a expression by RT–qPCR (normalized to actin expression) (n = 3–5 mice per group) (g) and SAβGal activity (h). i, Graph shows the number of SAβGal+ cells per mm2 in liver sections (n = 3 mice per group) and expression of Il1a, Il1b, Il6 and Mmp13 by RT–qPCR normalized to actin expression (n = 3–5 per group). j–n, Human CD34+ cord blood HPCs were transduced with BRAFV600E (BRAF-V600Ehu) or NGFR control (NGFRhu) lentiviral vectors, cultured in stem cell medium and analyzed for BrdU expression after daily pulses of BrdU from day 10 to day 13 (n = 4 different donors) (j), gene expression of senescence-associated genes using microarray expression profiling 7 d after transduction (n = 4 donors) (k) and SAβGal activity 9 d after transduction (l). l, The percentage of SAβGal+ cells among total cells is indicated (n = 4 donors). m, Trimethylation at lysine 9 of histone 3 (H3K9me3) and 4,6-diamidino-2-phenylindole (DAPI) immunofluorescence co-staining of NGFRhu and BRAF-V600Ehu HPCs 9 d after transduction; images are representative of senescence-associated-heterochromatin foci. n, IL-1α, IL-1β, IL-6 and IL-8 levels in the culture supernatant 7 d after HPC transduction (n = 4–6 donors). Data are represented as mean ± s.e.m.; statistical significance analyzed by unpaired and paired (for j,n) two-sided t-tests is indicated by *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Data are representative of at least two independent experiments.

Article Snippet: BRAF V600E or BRAF WT chimeras were generated by transplantation of 1–3 million whole BM cells flushed from the femurs of 8-week-old BRAF -V600E Scl + or BRAF -WT Scl + mice following tamoxifen injections into lethally irradiated (650 cGy × 2) CD45.1 mice aged 8–12 weeks (C57BL/6 background, Charles River Laboratories).

Techniques: Isolation, Injection, Irradiation, Transplantation Assay, Flow Cytometry, Control, Expressing, Purification, Real-time Polymerase Chain Reaction, Reverse Transcription, Quantitative RT-PCR, Activity Assay, Enzyme-linked Immunosorbent Assay, Transduction, Cell Culture, Gene Expression, Microarray, Immunofluorescence, Staining

Journal: Nature medicine

Article Title: BRAF V600E -induced senescence drives Langerhans cell histiocytosis pathophysiology

doi: 10.1038/s41591-021-01304-x

Figure Lengend Snippet: a,b, BRAF-V600Ehu-transduced CD34+ HPCs were cultured in stem cell medium and treated at day 1 and day 4 with rapamycin (10 nM) or dimethylsulfoxide (DMSO). a, Graphs show IL-1α and IL-1β protein levels in the supernatant of CD34+ HPCs 7 d after transduction (n = 4–5 independent donors). b, Percentages of MNPs (CD11c+ and/or CD14+) among total GFP+BRAF-V600E+ cells and among total GFP−BRAF-V600E− cells within cultures of BRAFV600E-transduced HPCs (BRAF-V600Ehu) that were treated with rapamycin or DMSO diluent were quantified by flow cytometry after 7 d of culture (n = 7 donors). c–e, BRAF-V600EScl+ mice were treated with rapamycin (0.5 mg per kg of body weight per day) or vehicle control for 10 d. Percentage of GMPs (defined as DAPI−lineage−c-Kit+Sca−CD16/CD32+CD34hi/int) among lineage-negative BM cells (c) and percentages of dendritic cells, macrophages and neutrophils among total CD45+ BM cells were measured using flow cytometry (n = 3–4 mice) (d). e, Liver weight in each group (n = 3–4 mice). Data are representative of two experiments. f, The percentage of tissue infiltration in the liver and lung is shown with representative images of H&E staining of tissues isolated from BRAF-V600EScl+ mice. g, Cartoon shows the experimental breeding scheme and treatment strategy used to generate BRAF-V600EScl+;ATTAC+ mice and control littermates. h,i, BRAF-V600EScl+;ATTAC+ mice and BRAF-WTScl+;ATTAC+ control littermates were treated with tamoxifen for 5 d and sacrificed 4 weeks later. h, The percentage of senescent p16INK4a-overexpressing cells was assessed by flow cytometry in the BM from BRAF-V600EScl+;ATTAC+ mice and control mice (n = 5–6 mice). Graphs show the percentage of GFP+ cells among live cells and among CD45+ cells, suggesting that senescent cells were exclusively hematopoietic cells. i, The nature of senescent p16INK4a-positive cells was assessed by flow cytometry. Graphs show the percentage of CD11b+ myeloid cells among GFP+ senescent cells and the percentage of macrophages, dendritic cells and neutrophils among GFP+CD11b+ myeloid senescent cells in BRAF-V600EScl+;ATTAC+ mice and control animals, highlighting that senescent cells in BRAF-V600EScl+;ATTAC+ mice are almost exclusively myeloid cells and that among them there is an accumulation of macrophages and dendritic cells, rather than neutrophils (n = 5–6 mice). j–l, Following tamoxifen injections, BRAF-V600EScl+;ATTAC+ mice and BRAF-WTScl+;ATTAC+ control littermates were treated with AP or vehicle for 3 weeks and sacrificed 4 weeks after tamoxifen injections. j, The percentage of senescent p16INK4a-overexpressing cells in the BM from untreated and treated BRAF-V600EScl+;ATTAC+ mice (n = 5) was assessed by flow cytometry. The graph shows the percentage of GFP+ cells among CD45+ cells. k, Liver weight in each group (n = 8). l, Percentages of tissue infiltration in the liver and lung are shown with representative images of H&E staining of tissues isolated from untreated and treated BRAF-V600EScl+;ATTAC+ mice (n = 8). Data are representative of three experiments and are represented as mean ± s.e.m.; statistical significance analyzed by unpaired and paired (for a,b) two-sided t-tests is indicated by *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Data are representative of at least two independent experiments.

Article Snippet: BRAF V600E or BRAF WT chimeras were generated by transplantation of 1–3 million whole BM cells flushed from the femurs of 8-week-old BRAF -V600E Scl + or BRAF -WT Scl + mice following tamoxifen injections into lethally irradiated (650 cGy × 2) CD45.1 mice aged 8–12 weeks (C57BL/6 background, Charles River Laboratories).

Techniques: Cell Culture, Transduction, Flow Cytometry, Control, Staining, Isolation

Journal: Nature medicine

Article Title: BRAF V600E -induced senescence drives Langerhans cell histiocytosis pathophysiology

doi: 10.1038/s41591-021-01304-x

Figure Lengend Snippet: Primary antibodies used for multiplexed immunohistochemical consecutive staining

Article Snippet: BRAF V600E or BRAF WT chimeras were generated by transplantation of 1–3 million whole BM cells flushed from the femurs of 8-week-old BRAF -V600E Scl + or BRAF -WT Scl + mice following tamoxifen injections into lethally irradiated (650 cGy × 2) CD45.1 mice aged 8–12 weeks (C57BL/6 background, Charles River Laboratories).

Techniques: Immunohistochemical staining, Incubation

Journal: The Journal of Molecular Diagnostics : JMD

Article Title: Frequency and Spectrum of BRAF Mutations in a Retrospective, Single-Institution Study of 1112 Cases of Melanoma

doi: 10.1016/j.jmoldx.2012.10.002

Figure Lengend Snippet: Profile of BRAF Mutations in 499 Cutaneous Melanomas, Including Primary and Metastatic Acral and Nonacral Lesions

Article Snippet: A : Pyrosequencing tracing shows the WT BRAF codon 600 as GTG.

Techniques:

Journal: The Journal of Molecular Diagnostics : JMD

Article Title: Frequency and Spectrum of BRAF Mutations in a Retrospective, Single-Institution Study of 1112 Cases of Melanoma

doi: 10.1016/j.jmoldx.2012.10.002

Figure Lengend Snippet: Detection of BRAF c.1799_1800delinsAA (the so-called p.V600E2) in melanoma by pyrosequencing and Sanger sequencing. A: Pyrosequencing tracing shows the WT BRAF codon 600 as GTG. B: In mutant BRAF codon 600, a prominent peak represents A after the first G (white arrow), and a reduced peak represents the third base G (black arrow). C: The precise variant sequence (GAA) is confirmed by Sanger sequencing, which demonstrates a heterozygous pattern of T/A G/A at the second and third base positions of codon 600 (arrows).

Article Snippet: A : Pyrosequencing tracing shows the WT BRAF codon 600 as GTG.

Techniques: Sequencing, Mutagenesis, Variant Assay