AmoyDx TM BRAF V600E Mutation Detection Kit

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1 AmoyDx TM BRAF V600E Mutation Detection Kit Detection of V600E mutation in the BRAF oncogene Instructions For Use Instructions Version: B3.1 Date of Revision: April 2012 Store at -20±2 o C 1/5

2 Background BRAF is a serine/threonine kinase that functions within the Ras-Raf-MEK-MAPK pathway. This pathway normally regulates cell proliferation and survival under the control of growth factors and hormones. Mutations in the BRAF gene have been associated with the development of cancer. The most common alteration in the BRAF gene is a mutation called V600E, which alters the valine at position 600 in the protein to a glutamic acid. The V600E mutation causes the BRAF protein to be permanently activated, even in the absence of growth factors. Aberrant BRAF signaling due to the V600E mutation can result in excessive cell proliferation and an adverse resistance to apoptosis. BRAF mutations occur in ~50% of melanoma tumors, ~40% of papillary thyroid tumors, ~30% of ovarian tumors, ~10% of colorectal tumors and ~10% of prostate tumors. The search for drugs that block oncogenic BRAF signaling is an active area of pharmaceutical research and development. The AmoyDx TM BRAF Mutation Detection Kit is highly selective and sensitive, detecting V600E mutation in the BRAF oncogene. AmoyDx s patented technology allows detection of 1% mutant DNA in a background of 99% normal DNA, while ensuring that false negatives are minimized. The procedure is easily adapted for use in high-throughput sample processing. Intended Use AmoyDx TM BRAF V600E Mutation Detection Kit is SFDA approved for clinical use in China and CE marked for IVD use in Europe. Kit Contents This kit contains sufficient reagents to carry out 24, 48 or 96 tests (Table 1), and additional mixed standard DNA (genomic DNA plus V600E mutated plasmid DNA) for positive control reactions. Table 1. Kit Contents Kit Size Reagents Supplied 24 reactions 48 reactions 96 reactions Volume Volume Volume #1: BRAF Reaction Mixture (1150 µl/ tube) 1 tube 2 tubes 4 tubes #2: BRAF Taq DNA Polymerase 20 µl 40 µl 80 µl #3: BRAF Mixed Standard (Positive Control) 250 µl 250 µl 250 µl Equipment and Reagents Not Supplied With Kit 1. Compatible PCR instruments are: Stratagene Mx3000P, Stratagene Mx3005P, ABI7300, ABI7500, ABI7900, ABI StepOne, LightCycler480 I and II, BioRad-CFX Sterile, nuclease-free tubes. 3. Dedicated pipette and filter pipette tips for handling DNA template. 4. Sterile, nuclease-free H2O. Shipping and Storage The kit requires cold-chain-transportation. The shelf-life of the kit is six months when the kit is stored immediately upon receipt at -20±2 o C in a constant-temperature freezer and protected from light. Specimen Material Human genomic DNA must be extracted from tissue or blood, or fixed paraffin-embedded tissue prior to use and stored at -20±2. Good DNA quality is essential and we recommend use of Qiagen DNA extraction kits (QIAamp DNA FFPE Tissue Kit, cat No , for paraffin embedded specimens; DNeasy Blood & Tissue kit, cat. No or 69506, for tissue and blood specimens). The OD value of DNA samples should be measured using the spectrophotometer after extraction. The Thermo Fisher NanoDrop 1000 /2000 spectrophotometer is recommended. Make sure A260/A230 value is greater than 2.0 and A260/A280 value between 1.8 and /5

3 Technological Principles The kit uses novel, proprietary primers and probes in a real-time PCR assay to detect the BRAF V600E mutation in human genomic DNA. The mutant BRAF V600E gene DNA is amplified by the specific primers, and detected by the novel probes. Protocol Notes: 1. Each reaction tube includes mutation detection system and internal control system. The mutation detection system is used to detect the mutation status of the BRAF gene (positive or negative). The internal control system is designed for detecting the presence of inhibitors, which may lead to false negative results. 2. The threshold at which the signal is detected above background fluorescence is called the Cycle threshold (Ct). The Ct values used to determine if a sample is positive or negative are based on extensive validation. If the Ct value falls within the appointed range (see below), the sample is classed as mutation positive. If the Ct value is outside the appointed range, the sample is classed as negative or below the detection limit of the kit. 3. The BRAF mixed standard contains a recombinant BRAF gene with the V600E mutation, and normal human genomic DNA. 4. It is recommended that the BRAF mixed standard should be analyzed during each PCR run, along with no-template controls. 5. The amount of sample DNA used for each sample PCR reaction depends on the kind of sample. a. For non-paraffin embedded samples, the recommended DNA amount in each test tube is 2 ~ 5 ng. i. Non-paraffin embedded specimens include fresh tissue, frozen pathological sections, non-heparin anticoagulant blood plasma, blood serum and non-heparin anticoagulant blood. b. For paraffin embedded samples with less than 3 years storage, we recommend use of 10 ng of template DNA. c. For paraffin embedded samples with more than 3 years storage, we recommend use of 15 ng of template DNA. d. The volume of DNA added to the reaction tube is 5 µl. Experimental Procedure 1. Thaw the BRAF Reaction Mixture. 2. Centrifuge BRAF Taq DNA polymerase and BRAF Reaction Mixture prior to use. 3. According to the ratio of 35 µl Reaction Mixture to 0.4 µl Taq DNA Polymerase per sample, transfer the appropriate amount of Reaction Mixture and Taq DNA Polymerase into a clean tube. 4. Mix the solution thoroughly by gently pipetting it up and down. a) Avoid vortexing solutions with Taq. 5. Centrifuge briefly. 6. Transfer 35 µl of the solution into the appropriate PCR tubes. 7. Add 5 µl sample DNA (2 to 15 ng per reaction, see above), 5 µl BRAF mixed standard or 5 µl ddh 2O (no-template control) to the appropriate PCR tubes. The layout for 22 samples, a positive control and a no template control is shown in Table 2. Table 2 Plate Layout (example for 24 tests/kit) Code A Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 B Sample 9 Sample 10 Sample 11 Sample 12 Sample 13 Sample 14 Sample 15 Sample 16 C Sample 17 Sample 18 Sample 19 Sample 20 Sample 21 Sample 22 STD NTC 8. Seal the PCR tubes. 9. Spin the PCR tubes gently to collect the reagents at the bottom of wells. 3/5

4 NOTE: This spin step is essential for proper mixing of the reagents. 10. Place the PCR tubes into the real-time PCR instrument. 11. Carry out real-time PCR using the cycling conditions described in Table 3. For:ADx-BR01 ADx-BR02 ADx-BR03 Table 3 Cycling Parameters Temperature Time Cycles Stage min 1 Stage s 64 20s s Stage s 60 35s Data collection of FAM and HEX/VIC s Sample Data Analysis 1. The FAM signal indicates the mutation status of sample and the HEX/VIC signal indicates the internal control status. 2. Make sure that each well gives a HEX/VIC signal. If the HEX/VIC signal gives a positive result, then continue with the analysis. If the Ct value <13, it indicates that the DNA was overloaded, and the amount of DNA should be reduced. If the HEX/VIC signal assay failed (Ct > 30), it shows that the DNA template contains PCR inhibitors. In this case, the DNA should be re-extracted and the whole experiment should be carried out again. 3. Ensure the calibration fluorescence is unselected, and select single mutation detection for each tube accordingly. It is necessary to choose reaction wells for positive control, no-template reference and samples simultaneously. Then, users can adjust the Threshold of FAM amplification curve, and obtain the Ct value of mutant group. 4. The mixed standard FAM Ct value should be less than 20; variation may occur in the Ct value due to different threshold settings on different instruments. 5. If the sample FAM Ct value 28, the sample is classed as negative or below the detection limit of the kit. If the sample FAM Ct value <28, the sample is classed as mutation positive. 6. The figure below shows a positive (Figure 1) and a negative result (Figure 2). Mixed Standard Sample Non-template Control Figure 1. Curve of DNA with mutant BRAF gene. 4/5

5 Mixed Standard Non-template Control Sample Figure 2. Curve of DNA with wild-type BRAF gene. Warnings and Precautions 1. Please read the instruction carefully and become familiar with all components of the kit prior to use. 2. Do not exchange and mix up the kit contents with different batches. 3. The kit and its contents cannot be resold or modified for resale without the written approval of AmoyDx. 4. Using other sources of reagents is not recommended. Strictly distinguish the reagents from mixed standard to avoid contamination. Otherwise, false positive may be produced. 5. Do the experiments with attention to prevent exogenous DNA contamination to reagents. It is recommended that users have separate, dedicated pipettes and filter pipette tips to add DNA template and during the preparation of reagents. 6. To optimize the activity and performance, mixtures should always be protected from light to avoid photo bleaching. 7. Only trained professionals should use this kit. Please wear suitable lab coat and disposable gloves. The used kit should be disposed of properly. 8. AmoyDx grants the customer a non-exclusive and non-transferable license to use AmoyDx technologies. 9. AmoyDx assumes no responsibility for any errors that may appear in this document. The information in this document is subject to change. References 1. Dhomen N, Marais M, New insight into BRAF mutations in cancer. Current Opinion in Genetics and Development 17: Thomas NE, BRAF somatic mutations in malignant melanoma and melanocytic naevi. Melanoma Research 16(2): /5

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