AmoyDx JAK2 Mutation Detection Kit Detection of V617F mutation in the JAK2 oncogene Instruction for Use For Research Use Only Instruction Version: B2.6 Revision Date: October 2013 Store at -20±5 Xiamen, Fujian 361026, PR China
Background For: ADx-JA01-R The JAK family of non-receptor tyrosine kinases includes JAK1, JAK2, JAK3, and TYK2. Growth factors and cytokines can regulate gene transcription through JAK-dependent activation of signal transducer and activator of transcription (STAT). The JAK-STAT signal transduction pathway regulates cell proliferation, differentiation, apoptosis and immune regulation. Activation of JAK2 by mutation of the amino acid at position 617 (V617F) is associated with myeloproliferative disorders (MPD), including polycythemia vera (mutations found in 90% of cases), essential thrombocythemia (50%) and idiopathic myelofibrosis (50%). JAK2 mutation is the main diagnostic indicator of MPD. The AmoyDx JAK2 Mutation Detection Kit is a highly selective and sensitive assay for detecting the V617F mutation in the JAK2 oncogene. Our company 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 The AmoyDx JAK2 Mutation Detection Kit is intended for research use only. Kit Contents This kit contains sufficient reagents to carry out 24 tests (Table 1), and additional Positive Control DNA (genomic DNA plus V617F mutated plasmid DNA) for positive control reactions. Table1 Kit Contents Tube No. Reagents Supplied Volume (µl) 1 V617F Reaction Mix 1250 2 JAK2 Taq DNA Polymerase 20 3 JAK2 Positive Control 150 Equipment and Reagents Not Supplied With Kit 1. Compatible PCR instruments: Stratagene Mx3000P, Stratagene Mx3005P, ABI7300, ABI7500, ABI StepOnePlus, LightCycler480 I and II, Bio-Rad CFX96. 2. Sterile, nuclease-free tubes. 3. Dedicated pipettes and filtered pipette tips for handling DNA template. 4. Sterile, nuclease-free H 2 O. Shipping and Storage The kit requires cold-chain-transportation. All contents of the kit should be stored immediately upon receipt at -20±5 in the dark in a constant temperature freezer. Avoid unnecessary freezing and thawing of the kit contents. Stability The shelf-life of the kit is eight months when stored under the recommended conditions and in the original packaging. Do not use the kit after the stated expiry date. Specimen Material Human genomic DNA must be extracted from non-heparin blood prior to use and stored at -20±2. High quality DNA is essential and we recommend use of DNA extraction kit (AmoyDx Blood DNA Kit, Cat No. ADx-BL01, for blood specimen). The OD value of DNA samples should be measured using the spectrophotometer after extraction. The NanoDrop 1000 /2000 spectrophotometer is recommended. Make sure A 260 /A 280 value between 1.8 1/4
and 2.0. Technological Principles The kit uses novel, proprietary primers and probes in a real-time PCR assay to detect the JAK2 V617F mutation in human genomic DNA. The mutant JAK2 V617F gene DNA is amplified by the specific primers, and detected by the novel probes. Protocol 1. Each reaction tube includes a mutation detection system and internal control system. The mutation detection system is used to detect the mutation status of the JAK2 gene. 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 JAK2 Positive Control contains a recombinant JAK2 gene with the V617F mutation, and normal human genomic DNA. 4. The mutation assay for each sample and control assay must be analyzed within the same PCR run to avoid run-to-run variations in threshold settings. It is recommended that the JAK2 Positive Control (PC) should be analyzed during each PCR run, along with no-template control (NTC). Experimental Procedure 1. Thaw the V617F Reaction Mix and JAK2 Positive Control at room temperature. 2. Centrifuge JAK2 Taq DNA Polymerase and V617F Reaction Mix prior to use. 3. Add 0.4 µl JAK2 Taq DNA polymerase to 45 µl V617F Reaction Mix per sample. 4. Mix the solution by gently pipeting it up and down. Note: avoid vortexing solutions with Taq. 5. Centrifuge briefly. 6. Transfer 45 µl of the above mixed solution into the appropriate PCR tubes. 7. Add 5 µl sample DNA (1~2 ng/µl), 5 µl JAK2 Positive Control (PC) or 5 µl ddh 2 O (no-template control, NTC) to the appropriate PCR tubes. 8. Seal the PCR tubes. 9. Spin the PCR tubes gently to collect the reagents at the bottom of tubes. Note: this spin step is essential for proper mixing of the reagents. 10. Place the PCR tubes into the real-time PCR instrument. 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 1 2 3 4 5 6 7 8 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 PC NTC 11. Carry out real-time PCR using the cycling conditions described in Table 3. Note: make sure the total volume of solution in each well is 50 µl (45 µl reagents plus 5 µl template). 2/4
Table 3 Cycling Parameters For: ADx-JA01-R Stage Temperature Time Cycles 1 95 5min 1 95 25s 2 60 20s 15 72 20s 93 25s 3 56 35s Data collection of FAM and HEX/VIC 26 72 20s Note: the probe settings on ABI machine: Reporter Dye: FAM, VIC; Quencher Dye: TAMRA; Passive Reference: NONE. Sample Data Analysis 1. The FAM signal indicates the mutation status of sample and the HEX/VIC signal indicates the internal control status. The HEX/VIC control amplifies and detects a region of genomic DNA adjacent to the JAK2 gene. 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 is overloaded, and the amount of DNA should be reduced. If the HEX/VIC signal fails, 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. Select the sample and controls as a group. Then adjust the threshold for FAM amplification curve to obtain the Ct value of the sample and controls. 4. The Positive Control FAM Ct value should be less than 21, but variation may occur due to different threshold settings on different instruments. 5. Analysis of mutation assay results. Check the FAM Ct value for each sample. Based on different mutant Ct values, the detection results are divided into strong positive, weak positive or negative. (1) Negative: if the sample FAM Ct value 26 (the critical negative value), the sample is classified as negative or below the detection limit of the kit. (2) If the FAM Ct value < 26 (the critical negative value), please analyze the results by the following approaches: a) Strong Positive: If the sample FAM Ct value is < 23 (critical positive value), the sample is classified as strong positive. b) If the sample FAM Ct value is 23 (critical positive value), the Ct of the reaction well shall be calculated to confirm the result. If the Ct value is < 5, the sample is confirmed as weak positive. If the Ct value 5, the sample is classified as negative or below the detection limit of the kit. c) The calculation of Ct: Ct = mutant FAM Ct value internal control HEX/VIC Ct value. The mutant FAM Ct value indicates the Ct value of the sample s FAM signal, the internal control Ct value indicates the Ct value of the sample s HEX/VIC signal. Warnings and Precautions 1. Please read the instruction carefully and become familiar with all components of the kit prior to use. 2. The product specified above does not contain any virus, reagent by-product of the same or metabolic by-product of Hepatitis A, B, C, D or HIV. 3. Do not exchange and mix up the kit contents with different batches. 3/4
4. The kit and its contents cannot be resold or modified for resale without the written approval of manufacturer. 5. Using other sources of reagents is not recommended. Strictly distinguish the reagents from Positive Control to avoid contamination. Otherwise, false positives may be produced. 6. Do the experiments with attention to prevent exogenous DNA contamination to reagents. It is recommended that users have separate, dedicated pipettes and filtered pipette tips to add DNA template during the preparation of reagents. 7. To optimize the activity and performance, mixtures should always be protected from light to avoid photo bleaching. 8. Only trained professionals could use this kit. Please wear suitable lab coat and disposable gloves. The used kit should be disposed of properly. Notes 1. Symbol for "KEEP DRY" 2. Symbol for "THIS WAY UP" 3. Symbol for "FRAGILE,HANDLE WITH CARE" References 1. Goldin LR, et al. Genome Med. 2009. 1:55. 2. Tefferi A, Leuk Lymphoma. 2008. 49:388-97. 3. Kilpivaara O, Nat Genet. 2009. 41: 455-9. 4/4