Primer probe composition, kit for detecting braf v600 gene mutation and application thereof

Abstract

The application discloses a primer probe composition for detecting a BRAF V600 gene mutation, a kit and application thereof. The application discloses a primer probe composition for detecting a BRAF V600 gene mutation, which comprises forward primers V600M-F and V600E-F shown in SEQ ID NO. 1 and SEQ ID NO. 2, a reverse primer V600R shown in SEQ ID NO. 3 and a detection probe V600P shown in SEQ ID NO. 4. Further disclosed are a kit containing the primer probe composition and application thereof. The primer probe composition is used for detecting a sample to be detected, has high sensitivity and specificity, can detect multiple BRAF V600 gene mutations, and can directly determine whether there is a mutation according to an amplification curve, without the need of calculating ΔCt, and is simple and direct.

Description

Technical Field

[0001] This invention relates to the field of biotechnology. More specifically, it relates to primer and probe compositions, kits, and applications for detecting BRAF V600 gene mutations. Background Technology

[0002] The BRAF gene is located on human chromosome 7q34. Its functional coding region consists of 2510 base pairs and encodes a serine / threonine protein kinase in the MAPK pathway. This enzyme transduces signals from RAS to MEK1 / 2, thereby participating in the regulation of various intracellular biological events. The most common BRAF gene mutation in cancer patients is the V600E mutation in exon 15, with BRAF V600K, V600R, V600D, and V600M accounting for approximately 10–20%. The BRAF V600E mutation can occur in various tumors, such as melanoma, colorectal cancer, thyroid cancer, and lung cancer. The NCCN guidelines recommend BRAF gene mutation testing in cases of DNA mismatch repair protein deficiency as part of the Lynch syndrome detection strategy. BRAF is the most common gene mutation in papillary thyroid carcinoma, and the BRAF V600E mutation can be used for the differential diagnosis of papillary thyroid carcinoma. More importantly, phase II clinical trials have demonstrated that melanoma patients carrying the BRAF V600E mutation respond to the BRAF kinase inhibitor vemurafenib with an efficacy exceeding 50%, and the 6-month overall survival is 84%. Therefore, BRAF gene mutation testing has become an essential diagnostic tool for various cancers. Existing BRAF V600 gene mutation detection technologies mainly include qPCR, ARMS, ddPCR, and NGS. While ddPCR and NGS offer good clinical value, their widespread clinical application has been limited due to the need for expensive equipment and cumbersome procedures. Currently, the main product for BRAF V600 mutation detection on the market is ARMS-PCR, which has the following disadvantages: (1) For single-point mutation detection, additional mutations need to be introduced at the 3' end of the primer or primers need to be self-paired to form a neck loop structure, which leads to reduced sensitivity. In clinical practice, there are often too few tumor cells in the puncture tissue of tumor patients. For the detection of point mutations in such samples, higher sensitivity technology is required to avoid the risk of false negatives caused by reagent sensitivity issues; (2) The specificity is low and it cannot completely inhibit the amplification of wild-type samples. The result interpretation requires the calculation of ΔCt, which is complicated and cannot meet clinical requirements. (3) It can only detect BRAF V600E gene mutations. Although p.V600E mutation is the most common mutation in the BRAF gene, accounting for 80-90% of all V600 codon mutations, other less common V600 codon mutations include: c.1798_1799delGTinsAA, p.V600K (5-12%), c.17981799delGTinsAG, p.V600R (~5%), c.1799_1800delTGinsAT, p.V600D (~1%), c.1798G>A, p.V600M (<1%), which also account for 10-20%, making it easy to miss detection.

[0003] Therefore, it is necessary to develop a new AMRS-PCR detection system for detecting BRAF V600 gene mutations to solve the above problems. Summary of the Invention

[0004] One object of the present invention is to provide a primer-probe composition with high sensitivity and specificity for detecting BRAF V600 gene mutations.

[0005] Another object of the present invention is to provide a kit for detecting BRAF V600 mutations comprising the above-described primer-probe composition and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The present invention first provides a primer and probe composition for detecting BRAF V600 gene mutations, comprising forward primers V600M-F and V600E-F as shown in SEQ ID NO.1 and SEQ ID NO.2, reverse primer V600R as shown in SEQ ID NO.3, and detection probe V600P as shown in SEQ ID NO.4.

[0008] Furthermore, the 5' end of the detection probe V600P is labeled with a fluorescent group selected from FAM, JOE, HEX, VIC, CY5, and TET, and the 3' end is labeled with a quenching group selected from TAMRA, MGB, and BHQ1.

[0009] Furthermore, the primer-probe composition further includes the forward internal standard primer GF as shown in SEQ ID NO.5, the reverse internal standard primer GR as shown in SEQ ID NO.6, and the internal standard probe GP as shown in SEQ ID NO.7.

[0010] Furthermore, the 5' end of the internal standard probe GP is labeled with a fluorescent group selected from FAM, JOE, HEX, VIC, CY5, and TET, and the 3' end is labeled with a quenching group selected from TAMRA, MGB, and BHQ1.

[0011] The present invention further provides the use of the above-described primer-probe composition in the preparation of reagents or kits for detecting BRAF V600 gene mutations.

[0012] The present invention also provides a kit for detecting BRAF V600 gene mutations, comprising the primer and probe composition described above for detecting BRAF V600 gene mutations.

[0013] Furthermore, the kit also includes other reagents required to complete the AMRS-PCR reaction. These reagents can be purchased or formulated according to methods described in the literature as needed by those skilled in the art. For example, the kit also includes some or all of the following: ARMS-specific Taq enzyme, real-time PCR reaction buffer, dNTPs, negative control, and positive control.

[0014] Furthermore, the ARMS-specific Taq enzyme is SNUPP Taq DNA polymerase, purchased from Shanghai Little Turtle Biotechnology Co., Ltd.; the real-time PCR reaction buffer is 5×SNUPP Taq Buffer; the negative control is nucleic acid-free water and / or 293T cell gDNA; and the positive control is a recombinant plasmid containing the BRAF V600 gene mutation.

[0015] The present invention further provides a method for detecting BRAF V600 mutations using the above-described primer-probe composition or kit, the method comprising the following steps:

[0016] (1) Obtain the genomic DNA (gDNA) of the sample to be tested;

[0017] (2) Using genomic DNA, negative control, or positive control as templates, construct a PCR amplification reaction system using the above primer and probe combination or kit, perform quantitative PCR amplification reaction, collect fluorescence signals, and observe the amplification curve;

[0018] (3) Results Analysis:

[0019] ① Positive: When the negative control has no amplification curve and the positive control has an obvious amplification curve, and the amplification curve of the sample to be tested has an obvious exponential growth phase, it indicates that the sample to be tested has a BRAF V600 gene mutation.

[0020] ② Negative: When the negative control has no amplification curve and the positive control has an obvious amplification curve, and the test sample has no Ct value and the curve has no obvious exponential growth phase, it indicates that the test sample does not have BRAF V600 gene mutation or the mutation rate of the test sample is lower than the minimum detection limit.

[0021] Furthermore, the total volume of the PCR amplification reaction system is 50 μL, comprising: 10 μL of 5×SNUPP Taq Buffer, 1 μL of 10 μM V600M-F, 1 μL of 10 μM V600E-F, 0.5 μL of 10 μM V600R, 1 μL of 2 μM GF, 1 μL of 2 μM GR, 0.5 μL of 2 μM GP, 1 μL of 5 U / μL SNUPP Taq DNA polymerase, 10-50 ng of genomic DNA or negative or positive control, and the remainder being ddH2O. See the table below for details:

[0022]

[0023]

[0024] Furthermore, the PCR amplification reaction program is as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 20 s, 64℃ annealing for 25 s, 68℃ extension for 25 s, 10 cycles; 95℃ denaturation for 15 s, 60℃ annealing for 20 s, 68℃ extension for 20 s, 35 cycles.

[0025] Furthermore, the BRAF V600 gene mutation includes one or more of the following: BRAF V600E (V600E1, V600E2), V600K, V600R, V600D (V600D1, V600D2), and V600M gene mutations.

[0026] The beneficial effects of this invention are as follows:

[0027] This invention optimizes and screens a large number of primers, probes, and Taq enzymes, ultimately obtaining a primer-probe composition for detecting BRAF V600 gene mutations and an ARMS-specific Taq enzyme that significantly improves detection sensitivity and specificity. Using this primer-probe composition and ARMS-specific Taq enzyme to detect samples, the sensitivity reaches 0.01%, requiring only 10 ng of gDNA from the sample, comparable to ddPCR. It simultaneously detects mutations in BRAF V600E1, V600E2, V600K, V600R, V600D1, V600D2, and V600M genes; it exhibits high specificity and can tolerate 500 ng of wild-type genome; the presence or absence of mutations can be directly determined from the amplification curve without calculating ΔCt, making result interpretation simple and direct.

[0028] The method for detecting BRAF V600 gene mutations in this invention is convenient, fast, and inexpensive, making it suitable for widespread application and providing rapid and accurate auxiliary medication guidance for clinical samples. It is especially advantageous for clinical prognostic observation and detection of samples with minimal lesions. Attached Figure Description

[0029] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0030] Figure 1 This study compares the sensitivity of three sets of primer-probe combinations.

[0031] Figure 2 This study investigated the blocking effects of three Taq enzymes.

[0032] Figure 3 Specific detection results for the BRAF V600 gene mutation detection kit

[0033] Figure 4 The results are for detecting BRAF V600E gene mutation samples prepared by mixing BRAF V600E gene mutation gDNA standard and wild-type 293T cell gDNA in a certain proportion using the method of the present invention.

[0034] Figure 5 The results are for detecting BRAF V600K gene mutation samples prepared by mixing BRAF V600K gene mutation gDNA standard and wild-type 293T cell gDNA in a certain proportion using the method of the present invention.

[0035] Figure 6 The results of digital PCR genotyping of 2182 clinical samples of colorectal cancer tumor tissue.

[0036] Figure 7 The results of digital PCR genotyping of 2271 clinical samples of colorectal cancer tumor tissue.

[0037] Figure 8 The results of digital PCR genotyping of 2151 clinical samples of melanoma tumor tissue. Detailed Implementation

[0038] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further explains the invention. Similar components in the drawings are indicated by the same reference numerals. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1: Establishment of a method for detecting BRAF V600 gene mutations

[0040] I. Screening of primer and probe compositions for BRAF V600 gene mutation detection

[0041] Through the design of numerous primers and probes, including linear primer-probe compositions (Table 1), circular primer-probe compositions (Table 2), and semi-circular primer-probe compositions (Table 3), the primer-probe compositions were optimized and screened. Finally, a semi-circular primer-probe composition that significantly improved detection sensitivity and specificity was obtained. The specific steps are as follows:

[0042] Table 1. Sequences of linear primer-probe compositions

[0043]

[0044] Table 2. Circulated primer-probe composition sequences

[0045]

[0046] Table 3. Sequences of semi-circular primer-probe compositions

[0047]

[0048]

[0049] gDNA was extracted from wild-type 293T cells. gDNA extracted from FFPE thyroid papillary carcinoma samples (confirmed to have BRAF V600E gene mutation using a commercially available ARMS-PCR BRAF V600E detection kit, purchased from Xiamen AIDE Biotechnology Co., Ltd., catalog number: 8.01.0151) was mixed with wild-type 293T cell gDNA at appropriate ratios, making the gDNA from clinical FFPE samples account for 0.01%, 0.1%, 0.5%, 1%, and 5% of the total DNA, respectively. Using this mixture as a template, a PCR amplification reaction system was constructed, and quantitative real-time PCR amplification was performed. Fluorescence signals were collected, and the amplification curves were observed to detect the sensitivity of three sets of primers and probes (linear primers and probes, circular primers and probes, and semi-circular primers and probes). ddH2O (NTC non-template control) and wild-type 293T cell gDNA were also set up as negative controls.

[0050] The PCR amplification reaction system is as follows:

[0051]

[0052] PCR amplification was performed using a real-time quantitative PCR instrument (SLAN-96P). The PCR amplification reaction process...

[0053] The order is:

[0054] Pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 20 s, annealing at 64℃ for 25 s, extension at 68℃ for 25 s, 10 cycles; denaturation at 95℃ for 15 s, annealing at 60℃ for 20 s, extension at 68℃ for 20 s, 35 cycles.

[0055] 15. Test results as follows Figure 1 As shown, the concentrations of linear primers and probes and circularized primers and probes were 0.1%.

[0056] The 0.5%, 1%, and 5% concentrations of BRAF V600E gene-mutated FFPE samples all showed excellent detection performance, and no amplification curves were observed for the negative controls of ddH2O and wild-type 293T cell gDNA, indicating that the sensitivity of the linear primers and probes, as well as the circular primers and probes, reached 0.1%. However, no amplification curve was observed for the 0.01% concentration, indicating that the sensitivity of the linear primers and probes, as well as the circular primers and probes, did not reach 0.01%. The semi-circular primers and probes all showed excellent detection performance for BRAF V600E-mutated FFPE samples at concentrations of 0.01%, 0.1%, 0.5%, 1%, and 5%, and no amplification curves were observed for the negative controls of ddH2O and wild-type 293T cell gDNA, indicating that the sensitivity of the semi-circular primers and probes reached 0.01%. Therefore, semi-circular primers and probes with significantly improved detection sensitivity were finally obtained, and their specific sequences are shown in Table 3.

[0057] II. Restrictive Effect Tests of Three Taq Enzymes

[0058] gDNA was extracted from wild-type 293T cells and diluted to 150 ng / µl, 75 ng / µl, 10 ng / µl, and 2 ng / µl as templates. Using the semi-circular primers and probes described in Table 3, and three Taq enzymes (ARMS-specific Taq enzyme SNUPP Taq DNA polymerase (purchased from Shanghai Xiaohaigui Biotechnology Co., Ltd., abbreviated as enzyme A), ARMS-specific Taq enzyme ARMS qPCR Mix multiplex mutation amplification arrest fluorescence quantitative premix (purchased from Shanghai Yisheng Biotechnology Co., Ltd., abbreviated as enzyme B), and non-ARMS-specific Taq enzyme Gold Multiplex PCR Mix (purchased from Kangwei Century Biotechnology Co., Ltd., abbreviated as enzyme C), PCR amplification reactions were constructed. Fluorescence signals were collected and amplification curves were observed to verify the 3' mismatch recognition ability of the three Taq enzymes. Simultaneously, recombinant plasmids containing BRAF V600E and V600M gene mutations were set as positive controls, and ddH2O (NTC non-template control) was used as a negative control.

[0059] The larger the Ct value, the stronger the blocking effect, as shown in the following results. Figure 2 As shown, enzyme C is the least effective, followed by enzyme B. Enzyme A has a stronger ability to recognize 3' mismatches and is more suitable for detecting BRAF V600 gene mutations in ARMS-qPCR.

[0060] III. Establishment of a BRAF V600 Gene Mutation Detection Kit

[0061] The BRAF V600 gene mutation detection kit includes a BRAF V600 gene mutation detection primer and probe composition (as shown in Table 3, including forward primers V600M-F and V600E-F as shown in SEQ ID NO.1 and SEQ ID NO.2, reverse primer V600R as shown in SEQ ID NO.3, and detection probe V600P as shown in SEQ ID NO.4, wherein the 5' end of detection probe V600P is labeled with the fluorescent group FAM and the 3' end is labeled with the quencher group BHQ1); forward internal standard primer GF and reverse internal standard primer GR as shown in SEQ ID NO.5 and SEQ ID NO.6, internal standard probe GP as shown in SEQ ID NO.7, wherein the 5' end of internal standard probe GP is labeled with the fluorescent group VIC and the 3' end is labeled with the quencher group BHQ1, real-time PCR reaction buffer (5×SNUPP Taq Buffer), dNTPs, and ARMS-specific Taq enzyme (SNUPP Taq DNA). The kit includes a polymerase and also contains two negative controls (ddH2O and wild-type 293T cell gDNA) and a positive control (recombinant plasmids containing mutations in the BRAF V600E1, V600E2, V600K, V600R, V600D1, V600D2, and V600M genes). Multiple quality controls help avoid false positives and false negatives.

[0062] IV. Establishment of a method for detecting BRAF V600 gene mutations

[0063] 1) Extract gDNA from the sample to be tested;

[0064] 2) Construct a PCR amplification reaction system using gDNA as a template, perform quantitative real-time PCR amplification, collect fluorescence signals, and observe the amplification curve;

[0065] The PCR amplification reaction system is as follows:

[0066]

[0067] PCR amplification was performed using a real-time quantitative PCR instrument (SLAN-96P / LightCycler480). The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 20 s, 64℃ annealing for 25 s, 68℃ extension for 25 s, 10 cycles; 95℃ denaturation for 15 s, 60℃ annealing for 20 s, 68℃ extension for 20 s, 35 cycles.

[0068] 3) Results Analysis:

[0069] ① Positive: When the negative control has no amplification curve and the positive control has an obvious amplification curve, and the amplification curve of the sample to be tested has an obvious exponential growth phase, it indicates that the sample to be tested has a BRAF V600 gene mutation.

[0070] ② Negative: When the negative control has no amplification curve and the positive control has an obvious amplification curve, and the test sample has no Ct value and the curve has no obvious exponential growth phase, it indicates that the test sample does not have BRAF V600 gene mutation or the mutation rate of the test sample is lower than the minimum detection limit.

[0071] Example 2: BRAF V600 gene mutation detection specificity test

[0072] Genomic DNA (gDNA) was extracted from 11 wild-type 293T cells and 72 whole blood samples from healthy voluntary blood donors using the Whole Blood DNA Extraction Kit from Zhongyuan Biotechnology Co., Ltd., following the kit's instruction manual. Specific detection was performed according to step four of Example 1, the BRAF V600 gene mutation detection method. ddH2O (NTC non-template control) was used as a negative control, and a recombinant plasmid containing the BRAF V600 gene mutation was used as a positive control.

[0073] Test results as follows Figure 3 As shown, the BRAF V600 gene mutation detection method of this invention detected fluorescence signals in the internal reference gene of 72 whole blood samples from healthy blood donors and 11 wild-type 293T cells. In addition, only the positive control showed fluorescence signals of the BRAF V600 mutant gene, while the 72 whole blood samples from healthy blood donors, 11 wild-type 293T cells and ddH2O showed no fluorescence signals, further demonstrating the specificity of the BRAF V600 gene mutation detection method of this invention.

[0074] Example 3: Sensitivity Detection of BRAF V600E and V600K Gene Mutations

[0075] 1. Sensitivity test for BRAF V600E gene mutation detection

[0076] The BRAF V600E gene mutation gDNA standard (30 ng / μL) containing 5% BRAF V600E gene mutation was purchased from Qinglang Gene Company and diluted with TE buffer to a concentration of 0.33% (2 ng / μL) to obtain BRAF V600E gene mutation samples. These samples were further diluted with 2 ng / μL 293T cell gDNA to obtain BRAF V600E gene mutation samples with concentrations of 0.1%, 0.05%, and 0.01%. Sensitivity testing was performed according to step four of Example 1, the BRAF V600 gene mutation detection method. ddH2O (NTC non-template control) and wild-type 293T cell gDNA were used as negative controls.

[0077] Test results as follows Figure 4 As shown, the BRAF V600 gene mutation detection method of the present invention has a good detection effect on BRAF V600E gene mutation samples with concentrations of 0.01%, 0.05%, 0.1%, and 0.33%, and does not have an amplification curve for ddH2O and wild-type 293T cell gDNA, indicating that the sensitivity of the method of the present invention reaches 0.01%.

[0078] 2. Sensitivity test for BRAF V600K gene mutation detection

[0079] A BRAF V600K gene mutation gDNA standard (30 ng / μL) containing 100% BRAF V600K gene mutation was purchased from Qinglang Gene Company. This standard was further diluted with 2 ng / μL 293T cell gDNA to prepare BRAF V600K gene mutation samples at concentrations of 5%, 1%, 0.1%, 0.05%, and 0.01%. Sensitivity testing was performed according to step four of Example 1, the BRAF V600 gene mutation detection method. ddH2O (NTC non-template control) and wild-type 293T cell gDNA were used as negative controls.

[0080] Test results as follows Figure 5 As shown, the BRAF V600 gene mutation detection method of the present invention has a good detection effect on BRAF V600K gene mutation samples with concentrations of 0.01%, 0.1%, 0.5%, 1%, and 5%, and does not show amplification curves for ddH2O and wild-type 293T cell gDNA, indicating that the sensitivity of the method of the present invention reaches 0.01%.

[0081] Example 4 Comparison with commercially available kits

[0082] Example 1, Step 4: BRAF V600 gene mutation detection method and commercial ARMS-PCR kit (purchased from Xiamen AIDE Biotechnology Co., Ltd., catalog number: 8.01.0151) were used to perform parallel detection on 545 clinical samples (including 58 cases of thyroid cancer, 400 cases of colorectal cancer, 81 cases of lung cancer, and 6 cases of melanoma).

[0083] The test results are shown in Table 4, the analysis table of clinical sample test results:

[0084] Table 4 Analysis of Clinical Sample Testing Results

[0085]

[0086] In a comparative experiment involving 545 clinical samples, the overall concordance rate between the BRAF V600 gene mutation detection method of this invention and the commercial ARMS-PCR kit was 99.45%. Three clinical samples showed discrepancies: the commercial ARMS-PCR kit detected negative results, while the method of this invention detected positive results. Further digital PCR confirmed that two colorectal cancer tissue samples (sample numbers 2182 and 2271) contained extremely low abundances of the BRAF V600E mutation, with mutation rates of 0.03% and 0.045%, respectively. (See details...) Figure 6 , Figure 7 One melanoma tissue sample (sample number 2151) showed a BRAF V600K mutation, which is outside the detection range of the commercial ARMS-PCR kit (which only detects BRAF V600E). See [link to kit]. Figure 8 This indicates that the detection method of the present invention has higher sensitivity and a wider detection range than commercial ARMS-PCR kits, which helps to reduce the clinical missed diagnosis rate.

[0087] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A primer-probe composition for detecting BRAF V600 gene mutations, characterized in that, The primer-probe composition includes forward primers V600M-F and V600E-F as shown in SEQ ID NO.1 and SEQ ID NO.2, reverse primer V600R as shown in SEQ ID NO.3, and detection probe V600P as shown in SEQ ID NO.4; The primer-probe composition further includes the forward internal standard primer GF as shown in SEQ ID NO.5, the reverse internal standard primer GR as shown in SEQ ID NO.6, and the internal standard probe GP as shown in SEQ ID NO.7; The BRAF V600 gene mutations include one or more of the following: BRAF V600E, V600K, V600R, V600D, and V600M gene mutations.

2. The primer-probe composition according to claim 1, characterized in that, The detection probe V600P is labeled with a fluorescent group at its 5' end and a quenching group at its 3' end. The internal standard probe GP is labeled with a fluorescent group at its 5' end and a quenching group at its 3' end. The fluorescent group is selected from one of FAM, JOE, HEX, VIC, CY5, and TET, and the quenching group is selected from one of TAMRA, MGB, and BHQ1.

3. The use of the primer-probe composition according to any one of claims 1-2 in the preparation of reagents for detecting BRAF V600 gene mutations, wherein the BRAF V600 gene mutations include one or more of the BRAF V600E, V600K, V600R, V600D and V600M gene mutations.

4. A kit for detecting BRAF V600 gene mutations, characterized in that, The kit includes the primer and probe composition according to any one of claims 1-2, wherein the BRAF V600 gene mutation includes one or more of the BRAF V600E, V600K, V600R, V600D, and V600M gene mutations.

5. The reagent kit according to claim 4, characterized in that, The kit also includes: ARMS-specific Taq enzyme, real-time PCR reaction buffer, dNTPs, negative control, and all of the positive control; the ARMS-specific Taq enzyme is SNUPTaq DNA polymerase; the negative control is nucleic acid-free water and / or 293T cell gDNA, and the positive control is a recombinant plasmid containing the BRAF V600 gene mutation.

Images