JAK2 MutaScreen TM Kit & Reference Scale

Transcription

1 For Life Science Research Use Only. Not Intended for Diagnostic Use. JAK2 MutaScreen TM Kit & Reference Scale Kit for detection of JAK2 V617F allele in human genomic DNA. Ref: MSPP-03 Tests: ABI Prism : 19 samples (in 1 experiment) in duplicate or 56 reactions LightCycler 2.0: LightCycler 480: Rotor-Gene TM 3000/6000: 14 samples (in 2 experiments) in duplicate or 64 reactions 20 samples (in 1 experiment) in duplicate or 56 reactions 19 samples (in 1 experiment) in duplicate or 56 reactions with a 72 wells rotor 10 samples (in 2 experiments) in duplicate or 56 reactions with a 36 wells rotor Note: Store Primers & Probes Mix in the dark and at -25 C to -15 C Control and Reference Scale at +4 C to +8 C Instructions for use Version 01, April First Edition. Page 1 of 28 DMK03-12

2 JAK2 MutaScreen TM & Reference Scale Kit for detection of JAK2 V617F allele in genomic DNA using either ABI Prism TaqMan, LightCycler or Rotor-Gene TM instruments. 1. Intended Use Background Technological Principle RQ-PCR TaqMan Allelic discrimination assay principle Reagents and Instruments Materials provided Handling and storage Kit stability Quality Control Warnings Reagents and material required Reagents Equipment Warnings and precautions Instructions for Use DNA Samples ABI Prism TaqMan or LightCycler 480 instruments LightCycler instrument (2.0) Corbett Rotor-Gene TM instruments 3000/ Detailed procedure ABI Prism TaqMan instruments LightCycler instrument (2.0) LC480 instrument Rotor-Gene TM instruments 3000/ Data Analysis Expression of the results Notice Contact information Appendix ABI Prism 7700 Instrument setting Preparing a New Plate Read File Running a PlateRead Plate Read Analysis Analyzing a Plate Read First Edition. Page 2 of 28 DMK03-12

3 1. Intended Use The JAK2 MutaScreen TM Kit is a research tool intended for the accurate detection of JAK2 V617F mutation and determine proportion of allele load in genomic DNA extracted from human cells. 2. Background The mutation affecting the Janus Tyrosine Kinase 2 (JAK2 V617F) was identified by the INSERM team of Dr. Vainchenker. This mutation is found in a large proportion of patients with PV, ET (Polycythemia Vera, Essential Thrombocythemia) and less frequently in IMF (chronic Idiopathic MyeloFibrosis) patients. This acquired mutation occurs in the pseudokinase domain of JAK2 in hematopoietic cells, and was shown to be responsible for the constitutive activation of molecular signalling pathways, leading to an uncontrolled cell proliferation in MPD (MyeloProliferative Disorders). JAK2 (9p24) V617F 1 kb 10 kb 14 G T Forward Primer Reverse Primer Allele Specific Probes Figure 1: Human JAK2 gene and position of the V617F point mutation on exon Technological Principle 3.1. RQ PCR Quantitative PCR data can be rapidly obtained without post-pcr processing by real-time detection of fluorescent signals during and/or subsequent to PCR cycling, thereby significantly reducing the risk of PCR product contamination. At present, three major types of RQ-PCR techniques are available: RQ-PCR analysis using i) SYBR Green I Dye, ii) hydrolysis probes and iii) hybridisation probes. This assay exploits the RQ-PCR Double Dye Hydrolysis Oligonucleotide Probes principle. Forward and reverse primers hybridise to a specific sequence product during PCR amplification. Double Dye probes are contained in the same mix. Those probes, which consist of an oligonucleotide labelled with a 5 reporter dye and a downstream, 3 quencher dye, hybridise to their target sequences within the PCR product. RQ-PCR analysis with hydrolysis probes exploits the 5'-3' exonuclease activity of the Thermus aquaticus (Taq) polymerase. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence primarily by Förster-type energy transfer. During PCR, if the target of interest is present, the probe specifically anneals between the forward and reverse primer sites. The 5-3 exonuclease activity of the DNA polymerase cleaves the probe between the reporter and the quencher only if the probe hybridises to the target. The probe fragments are then displaced from the target, and polymerisation of the strand continues. The 3 end of the probe is blocked to prevent its extension during PCR (see Figure 2). This process occurs in every cycle and does not interfere with the exponential accumulation of product. The increase in fluorescence signal is detected only if the target sequence is complementary to the probe and hence amplified during PCR. Because of these requirements, non-specific amplification is not detected. Thus, the increase in fluorescence is directly proportional to the target amplification during PCR TaqMan Allelic discrimination assay principle. In allelic discrimination assays, the PCR assay includes a specific, fluorescent, dye-labeled probe for each allele. The probes contain different fluorescent reporter dyes (FAM TM and VIC ) to differentiate the amplification of each allele. During PCR, each probe anneals specifically to complementary sequences between the forward and reverse primer sites. AmpliTaq Gold DNA polymerase can cleave only probes that hybridize to the allele. Cleavage separates the reporter dye from the quencher dye, which results in First Edition. Page 3 of 28 DMK03-12

4 increased fluorescence by the reporter dye. Thus, the fluorescence signal(s) generated by PCR amplification indicate(s) the alleles that are present in the sample. Mismatches between a probe and allele reduce the efficiency of probe hybridization. Furthermore, AmpliTaq Gold DNA polymerase is more likely to displace the mismatched probe rather than cleave it to release reporter dye. The figure below illustrates results from matches and mismatches between allele and probe sequences in allelic discrimination assays. Probe 1 Probe degradation Allele 1 Match Substantial increase of FAM Signal Probe 2 Allele 2 Match Probe degradation Substantial increase of VIC Signal Probe displacement Probe 1 Allele 2 Mismatch No Signal Probe displacement Probe 2 Allele 1 Mismatch No Signal Figure 2: Both specific probes contain a non-fluorescent quencher at the 3 end. Because this quencher does not fluoresce, the Sequence Detection Systems instruments can measure the reporter dye contributions more precisely A PC NC RS TS1 TS2 TS3 B PC NC RS TS1 TS2 TS3 C PC NC RS TS1 TS2 TS3 D E PS 01 PS 01 PS 05 PS 05 PS 09 PS 09 H2O F PS 02 PS 02 PS 06 PS 06 PS 10 PS 10 H2O G PS 03 PS 03 PS 07 PS 07 H2O H PS 04 PS 04 PS 08 PS 08 Amplification Run chap. 5.2 (TaqMan) or chap. 5.3 (LightCycler) chap. 8.2 (RotorGene) TS1 TS2 TS2 TS1 TS3 RS TS3 RS H2O H2O NC NC S01 PC S01 PC S02 S09 S02 S09 S03 S08 S03 S08 S04 S07 S04 S07 S06 S06 S05 S05 Post-Read Run chap (TaqMan) or chap (LightCycler) chap (RotorGene) Export Data Analysis chap. 5.5 First Edition. Page 4 of 28 DMK03-12

5 4. Reagents and Instruments 4.1. Materials provided Vial Name Content Volume Part Number V617F Positive Control PC-VF 100% VF Allele 30µl IP-PF V617F Negative Control NC-VF 0% VF Allele 30µl IP-PF Reference Scale M1 M1-VF 2% VF Allele Detection limit 30µl IP-PF Reference Scale M2 M2-VF 5% VF Allele 30µl IP-PF Reference Scale M3 M3-VF 12.5% VF Allele 30µl IP-PF Reference Scale M4 M4-VF 31% VF Allele 30µl IP-PF Reference Scale M5 M5-VF 50% VF Allele 30µl IP-PF Reference Scale M6 M6-VF 78% VF Allele 30µl IP-PF Primers and Probes Mix Content Volume Part Number PPM- JAK2 V617F 10X Amber tube Supplied ready-to-use Mix of specific reverse and forward primers for the JAK2 gene, specific V617F FAM TM probe and Wild Type VIC probe Note: Spin all the JAK2 Controls and the Primers and Probes tubes before use. 145µl IP-PF Handling and storage Note: kits are shipped at room temperature but PPM should be stored at -25 C to -15 C in a constanttemperature freezer immediately upon receipt. Keep the Primers & Probes Mix (PPM tube) away from light, as this product is photosensitive. Store Controls (PC-VF, NC-VF and Reference Scale) at +4 C to +8 C. Centrifuge the tubes before opening. Expiration dates for each reagent are indicated on the individual component labels. These storage conditions apply to both opened and unopened components. The product will maintain performance through the control date printed on the label. Exposure to light, heat or humidity may affect the shelf life of some of the kit components and should be avoided. Components stored under conditions other than those stated on the labels may not perform properly and may adversely affect the assay results. Store all unopened kit components in original containers Kit stability The product will maintain performance through the control date printed on the label under correct storage conditions Quality Control Quality control of the complete kit has been performed on an ABIPrism TaqMan Warnings The users must have been trained and familiar with this technology prior to the use of this device. Perform the test according to the Good Laboratory Practice (GLP) guidelines for PCR applications. First Edition. Page 5 of 28 DMK03-12

6 4.2. Reagents and material required Reagents Additional reagents to be provided by the user are: Buffer and Taq Polymerase (we recommend the use of the TaqMan Universal PCR Master Mix, Applied Biosystems or the specific LightCycler TaqMan Probe Master Mix) General laboratory equipment Specific material and reagent for LightCycler use Nuclease-free PCR grade H 2 O Recommended Validated Reagents Reagent Validated reagent (Brand name) Validated reagent (Provider, reference in EU) PCR Master Mix TaqMan Universal PCR Master Mix Applied Biosystems # Freshly Prepared 5X Master Mix Light Cycler TaqMan Probe Master kit Roche # Equipment To perform the assay, you should need the following equipment: Real-time PCR instrumentation (TaqMan ABI or any equivalent equipment) 0.5ml or 0.2ml RNase- and DNase free PCR tubes General laboratory equipment Biophotometer Nuclease free aerosol-resistant sterile PCR pipette tips with hydrophobic filters Sterile reaction cups (eppendorf) for preparing dilutions Microcentrifuge equipped for 0.2 ml/0.5ml tubes. Max speed: / rpm Microliter pipettor dedicated for PCR (1-10µl; µl; µl) Molecular biology grade water Note: This assay could be used on any Real-time PCR instrument, but it has been only formally validated for its use on TaqMan ABI 7000, 7700, 7900, LightCycler 2.0 and Warnings and precautions NOTE: The reagents and instructions supplied in this kit have been validated for optimal performance. Further dilution of the reagents or alteration of incubation temperatures may give erroneous or discordant results. Differences in sample processing and technical procedures in the user's laboratory may invalidate the assay results. Use nuclease-free labware (e.g. pipettes, tips, vials) and wear gloves when performing the assay. Use fresh aerosol-resistant tips for all pipetting steps to avoid cross-contamination. Transfer the required solutions for one experiment into a fresh tube to avoid carry-over contamination. Manipulate the Control DNA dilutions (PC-VF, NC-VF and Reference Scale) in a separate room. Minimise microbial contamination of reagents to avoid non-specific reactions. Incubation times, temperatures, or methods other than those specified might give erroneous results. Wear appropriate personal protective equipment to avoid contact with eyes and skin. Refer to the Materials Safety Data Sheet (MSDS) for additional information. Human tissues must be handled as if capable of transmitting infections and disposed of with proper precautions, and in compliance with OSHA and/or CAP (or EU equivalent) guidelines. Never pipette kit reagents by mouth and avoid contact with skin and mucous membranes. If reagents are exposed to sensitive areas, wash thoroughly with copious amount of water and contact a physician. All reagents provided are formulated specifically for use with this test. No substitutions should be made to obtain optimal performance of this assay. First Edition. Page 6 of 28 DMK03-12

7 5. Instructions for Use Before you begin: The user should read these instructions carefully and become familiar with all components prior to use DNA Samples Genomic DNA should be obtained either from purified PBL of whole blood, polynuclear cells or granulocytes. DNA extraction should be performed by any classical or commercial method (ex: QiAmp DNA mini kit Qiagen). The resultant DNA has to be diluted at 5ng/µl in TE PH8.0 and stored at +4 C to +8 C ABI Prism TaqMan or LightCycler 480 instruments Collect and thaw all necessary components and place them on ice. Spin briefly all the tubes (~10sec, 10,000rpm, to collect the liquid in the bottom of the tube) Prepare the following RQ-PCR premix according to the number of samples being processed. Each experiment should contain in duplicate, the positive and negative controls, a non-template control (H2O), all reference scale points and up to 19 samples to be tested. RQ-PCR premix Reagents Final Conc. 1 Reaction 56 Reactions 1 experiment TaqMan Universal PCR Master Mix 2X Applied Biosystems (Not Provided) 1X 12.5 µl 725 µl IPSOGEN Primers & Probes mix 10X 1X 2.5 µl 145 µl Adjust vol. to 20 µl with nuclease-free H 2 O 5 µl 290 µl Total volume = 20 µl Material to be quantified (Sample DNA, Control or Water) 5 µl All mentioned concentrations are for the final volume of the reaction. The above table describes the pipetting scheme for the preparation of one reagent mix, calculated to achieve a final PCR reaction volume of 25µl. A pre-mix can be prepared, according to the number of reactions. To be performed on ice: Dispense 20 µl of the RQ-PCR pre-mix per well. Add 5 µl of material to be quantified (25ng Sample genomic DNA or Controls or Reference Scale) in the corresponding well (total volume 25µl). Mix gently, by pipetting up and down. Place the plate in the thermal cycler A PC M1 M4 S 01 S 04 S 07 S10 S 13 S 16 S 19 B PC M1 M4 S 01 S 04 S 07 S 10 S 13 S 16 S 19 C D NC M2 M5 S 02 S 05 S 08 S 11 S 14 S 17 E NC M2 M5 S 02 S 05 S 08 S 11 S 14 S 17 F G H2O M3 M6 S 03 S 06 S 09 S 12 S 15 S 18 H H2O M3 M6 S 03 S 06 S 09 S 12 S 15 S 18 Suggested plate set-up for one single experiment. PC: Positive Control (provided); NC: Negative Control (provided); M: Reference Scale (provided); S: Unknown DNA Sample First Edition. Page 7 of 28 DMK03-12

8 Amplification Run: Run the following program: RQ-PCR program Temperature Time Cycles 50 C 2 min X 1 95 C 10 min X 1 92 C 15 sec 60 C 1 min Post-Read-Run: See below (Chap. # for ABI instruments and for LC480) LightCycler instrument (2.0) NB: Because of particular technological requirements, LightCycler experiments must be performed using specific reagents. We recommend the use of the Light Cycler TaqMan Probe Master kit and to follow the manufacturer's instructions to prepare the Master Mix 5X. Collect and thaw all necessary components and place them on ice. Spin briefly all the tubes (~10sec, 10,000rpm, to collect the liquid in the bottom of the tube) Prepare the following RQ-PCR premix according to the number of samples being processed. Each experiment should contain in duplicate, the positive and negative controls, a non-template control (H2O), all reference scale points and up to 7 samples to be tested. RQ-PCR premix Reagents Final Conc. 1 Reaction 32 Reactions 1 experiment Freshly prepared Master Mix 5X Light Cycler TaqMan Probe Master kit (Not Provided) 1X 4 µl 136 µl IPSOGEN Primers & Probes mix 10X 1X 2 µl 68 µl Adjust vol. to 15 µl with nuclease-free H 2 O 9 µl 306 µl Total volume = 15 µl Material to be quantified (Sample DNA, Control or Water) 5 µl All mentioned concentrations are for the final volume of the reaction. The above table describes the pipetting scheme for the preparation of one reagent mix, calculated to achieve a final PCR reaction volume of 20µl. A pre-mix can be prepared, according to the number of reactions. To be performed on ice: NC Dispense 15 µl of the RQ-PCR pre-mix per capillary. Add 5 µl of material to be quantified (25ng Sample genomic DNA or Controls or Reference Scale) in the corresponding capillary (total volume 20µl). Mix gently, by pipetting up and down. Load the samples in the apparatus according to the manufacturer recommendations. NC H2O H2O M1 M1 M2 M2 M3 M3 M4 M4 M5 X 50 PC PC M5 M6 S7 S7 S6 S6 S5 S5 S4 S4 S3 S3 S2 S1 S2 M6 S1 Suggested rotor set-up for each experiment. PC: Positive Control (provided) NC: Negative Control (provided) M: Reference Scale (provided) PS: Unknown DNA Sample First Edition. Page 8 of 28 DMK03-12

9 Amplification Run: Run the following PCR program: Post-Read-Run: See below (Chap. # 5.5.2). LightCycler RQ-PCR program for TaqMan Probe Temperature Time Cycles Ramp Acquisition 55 C 2 min. X 1 20 None 95 C 10 min. X 1 20 None 92 C 15 sec. 20 None X C 1 min 20 Single 5.4. Corbett Rotor-Gene TM instruments 3000/6000 Collect and thaw all necessary components and place them on ice. Spin briefly all the tubes (~10sec, 10,000rpm, to collect the liquid in the bottom of the tube) Prepare the following RQ-PCR premix according to the number of samples being processed. Each experiment should contain in duplicate, the positive and negative controls, a non-template control (H2O), all reference scale points and up to 19 samples (72 wells) ) or 5 samples (36 wells) to be tested. RQ-PCR premix Reagents Final Conc. 1 Reaction 56 Reactions 1 experiment TaqMan Universal PCR Master Mix 2X Applied Biosystems (Not Provided) 1X 12.5 µl 725 µl IPSOGEN Primers & Probes mix 10X 1X 2.5 µl 145 µl Adjust vol. to 20 μl with nuclease-free H2O 5 µl 290 µl Total volume = 20 µl Material to be quantified (Sample DNA, Control or Water) 5 µl All mentioned concentrations are for the final volume of the reaction. The above table describes the pipetting scheme for the preparation of one reagent mix, calculated to achieve a final PCR reaction volume of 25μl. A pre-mix can be prepared, according to the number of reactions. To be performed on the cooling block (provided with the Rotor-Gene TM ): Dispense 20 μl of the RQ-PCR pre-mix per tube. Add 5 μl of material to be quantified (25ng Sample genomic DNA or Controls or Reference Scale) in the corresponding tube (total volume 25μl). Mix gently, by pipetting up and down. Place the tubes on the appropriate rotor. Attach the locking ring on the rotor. Place the rotor in the Rotor-Gene TM. First Edition. Page 9 of 28 DMK03-12

10 H2O H2O M1 M1 M2 M2 PC PC S5 S5 S5 NC NC M3 M3 M4 M4 M5 M5 M6 M6 Example for experiment rotor set-up (36 wells Rotor). PC: Positive Control DNA (provided); NC: Negative Control DNA (provided); M: Reference Scale (provided); S: Unknown DNA Sample S4 S4 S1 S1 S3 S3 S2 S2 Amplification Run: Run the following program: Post-Read-Run: See below (Chap. # 5.5.4). RQ-PCR program Temperature Time Cycles 50 C 2 min X 1 95 C 10 min X 1 92 C 15 sec 60 C 1 min X 50 First Edition. Page 10 of 28 DMK03-12

11 5.5. Detailed procedure People who are familiar with the use of the function "allelic discrimination" on ABI can go directly to chapter 5.5 page ABI Prism TaqMan instruments. Reaction Plate ABI System Amplification Run Post-Read Run Note: Instrument setting could slightly differ between apparatus. Please refer to your user manual for more details. For the ABI 7700 instrument, please also refer to chap. 8.1 (appendix) below. 1. Select Start > Programs > 2. Select File > New. 3. In the New Document Wizard: a. Click the Assay drop-down list, then select Allelic Discrimination. b. Accept the default settings for the Container and Template fields (96-Well Clear and Blank document). c. In the Plate Name field, type AD Post-Read. AD Post-read 4. Click Next> to access the Select Markers page. If the Marker list in the Select Markers page contains a marker suitable for your application, skip to step 6. First Edition. Page 11 of 28 DMK03-12

12 5. If the Marker list does not contain a marker suitable for your application, as above, create detectors and marker: a. Click New Detector. b. In the New Detector dialog box, type Allele A for Name. c. Leave the Reporter Dye set to FAM TM. d. Click the color button, select a color, and then click OK. e. Click Create Another. f. For Name, type Allele B. g. Select VIC for the Reporter Dye. h. Click the color button, select a color, and then click OK. 6. In the Select Markers Window: a. Click New Marker. b. In the New Marker dialog box, type JAK2 for Name. c. Select the Allele A and Allele B detectors you created above. d. Click OK. 7. In the Select Markers window: Select either the JAK2 marker you created above or a suitable marker, then click Add >>. First Edition. Page 12 of 28 DMK03-12

13 Note: To remove a marker, select it, then click Remove. 8. Click Next >. 9. In the Setup Sample Plate page, select the marker for wells: Click-drag to select wells A1 through H Click Finish. 11. Select the Instrument tab. Change the Sample Volume to 25 µl. 12. Select File > Save, and then click Save to retain the name you assigned when you created the plate document. 13. Load the reaction plate into the instrument. 14. Start the amplification Run. 15. Click Post-Read. The instrument will performed a run of 1cycle, 60 sec. at 60 C. During this post-read run, the instrument collects one fluorescent scan per well. 16. Export results. Select File > Export, then click results. 17 Analyse export file. Ex: Fluorescence VIC Sample VIC 1 du sample 1 Fluorescence FAM FAM Sample du 1 sample 1 First Edition. Page 13 of 28 DMK03-12

14 LightCycler instrument (2.0) 1- At the end of the amplification Run, Select a new LightCycler experiment. 2 "Post read" step. Run the following PCR program: Temperature Time Cycles Ramp Acquisition 60 C 1 min. X 1 20 Single 3 On the window Online data display, click right near to the current data fluorescent graph: 4 Choose export. First Edition. Page 14 of 28 DMK03-12

15 5 On the Export Chart, choose Excel. 6 In the filename, choose the place to export your result file. 7 Click export. 8 Analyse export file. EX: Position Capillary VIC data FAM data First Edition. Page 15 of 28 DMK03-12

16 LC480 instrument 1- At the end of the amplification Run, Select a new experiment 2- "Post read" step Detection format: Multi Color Hydrolysis Probe Customize: Select channels FAM ( ) & Hex ( ) Volume: 25µl Run the following PCR program: Temperature Time Cycles Acquisition 60 C 1 min. X 1 Single Export FAM fluorescence 3- On the window Fluorescence History, click right and choose export : First Edition. Page 16 of 28 DMK03-12

17 5- On the Export Chart, choose XML 6- In the filename, choose the place to export your result file 7- Click export Export VIC fluorescence 8 Click on the button (near the red arrow) and select the channel. Start again the export at stage 3 * * 9- Analyse export file EX: Well position column Fluorescence data column First Edition. Page 17 of 28 DMK03-12

18 Rotor-Gene TM instruments 3000/ Fluorescence Cycle Note: Instrument setting could slightly differ between apparatus. Please refer to your user manual for more details. 1. Start the Rotor-Gene TM Software. In the New Run pop-up window select the Advanced tab and double click the Hydrolysis Probes option. 2. On the following window set the reaction volume to 25 μl and click Next. First Edition. Page 18 of 28 DMK03-12

19 3. Then on the next window click the Edit Profile button to create the PCR program. Be sure to add the last acquiring step at 60 C (one cycle) for both channels Green (FAM) and Yellow (VIC) so that the instrument takes the final Post-Run Read: 4. Please, also arrange the Auto-Gain feature to perform the calibration at 60 C for the Green and Yellow channels before 1 st acquisition as follows: First Edition. Page 19 of 28 DMK03-12

20 5. At the end of the run click File > Save As > Excel Date Sheet. 6. After naming the export a pop-up window appears. Click OΚ leaving the option disabled. The data from the Post-Run Read can be found under the Cycling B section in the Excel data sheet. First Edition. Page 20 of 28 DMK03-12

21 5.6. Data Analysis Whatever the instrument used you will obtain an Export File from which you can extract the following data: rn Fluo VIC rn Fluo FAM Positive Control 1,24 8,93 Positive Control 1,15 8,96 Negative Control 3,58 2,39 Negative Control 3,63 2,44 H2O 0,61 1,52 H2O 0,67 1,00 Reference Scale M1 3,62 2,91 Reference Scale M1 3,60 2,79 Reference Scale M2 3,68 3,49 Reference Scale M2 3,60 3,33 Reference Scale M3 3,69 4,59 Reference Scale M3 3,68 4,70 Reference Scale M4 3,63 6,34 Reference Scale M4 3,79 6,60 Reference Scale M5 3,86 7,72 Reference Scale M5 3,78 7,53 Reference Scale M6 3,07 8,26 Reference Scale M6 3,15 8,16 Sample 1 3,65 2,40 Sample 1 3,68 2,44 Sample 2 2,25 8,43 Sample 2 2,34 8,30 Sample 3 3,83 5,71 Sample 3 4,09 6,56 Sample 4 4,33 2,48 Sample 4 4,25 2,44 Sample 5 2,36 8,03 Sample 5 2,42 7, For each test, calculate the FAM TM /VIC ratio: rn Fluo VIC rn Fluo FAM Ratio FAM/VIC Positive Control 1,24 8,93 7,22 Positive Control 1,15 8,96 7,83 Negative Control 3,58 2,39 0,67 Negative Control 3,63 2,44 0,67 H2O 0,61 1,52 H2O 0,67 1,00 Reference Scale M1 3,62 2,91 0,80 Reference Scale M1 3,60 2,79 0,78 Reference Scale M2 3,68 3,49 0,95 Reference Scale M2 3,60 3,33 0,92 Reference Scale M3 3,69 4,59 1,24 Reference Scale M3 3,68 4,70 1,28 Reference Scale M4 3,63 6,34 1,75 Reference Scale M4 3,79 6,60 1,74 Reference Scale M5 3,86 7,72 2,00 Reference Scale M5 3,78 7,53 1,99 Reference Scale M6 3,07 8,26 2,69 Reference Scale M6 3,15 8,16 2,59 Sample 1 3,65 2,40 0,66 Sample 1 3,68 2,44 0,66 Sample 2 2,25 8,43 3,74 Sample 2 2,34 8,30 3,55 Sample 3 3,83 5,71 1,49 Sample 3 4,09 6,56 1,61 Sample 4 4,33 2,48 0,57 Sample 4 4,25 2,44 0,58 Sample 5 2,36 8,03 3,41 Sample 5 2,42 7,59 3, First Edition. Page 21 of 28 DMK03-12

22 2-Then, calculate the mean ratio for each sample (Control and Unknown samples): rn Fluo VIC rn Fluo FAM Ratio FAM/VIC Mean Ratio Positive Control Negative Control 1,24 3,58 8,93 2,39 7,22 0,67 Positive Control Negative Control 1,15 3,63 8,96 2,44 7,83 0,67 7,52 0,67 H2O 0,61 1,52 H2O 0,67 1,00 Reference Scale M1 Reference Scale M2 Reference Scale M3 Reference Scale M4 Reference Scale M5 Reference Scale M6 Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 3,62 3,68 3,69 3,63 3,86 3,07 3,65 2,25 3,83 4,33 2,36 2,91 3,49 4,59 6,34 7,72 8,26 2,40 8,43 5,71 2,48 8,03 0,80 0,95 1,24 1,75 2,00 2,69 0,66 3,74 1,49 0,57 3,41 Reference Scale M1 Reference Scale M2 Reference Scale M3 Reference Scale M4 Reference Scale M5 Reference Scale M6 Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 3,60 3,60 3,68 3,79 3,78 3,15 3,68 2,34 4,09 4,25 2,42 2,79 3,33 4,70 6,60 7,53 8,16 2,44 8,30 6,56 2,44 7,59 0,78 0,92 1,28 1,74 1,99 2,59 0,66 3,55 1,61 0,58 3,14 0,79 0,94 1,26 1,74 1,99 2,64 0,66 3,65 1,55 0,57 3, Simply compare the mean ratio value obtained for Unknown Samples with Reference Scale mean ratio values. rn Fluo VIC rn Fluo FAM Ratio FAM/VIC Mean Ratio Positive Control Negative Control 1,24 3,58 8,93 2,39 7,22 0,67 Positive Control Negative Control 1,15 3,63 8,96 2,44 7,83 0,67 7,52 0,67 H2O 0,61 1,52 H2O 0,67 1,00 Reference Scale M1 Reference Scale M2 Reference Scale M3 Reference Scale M4 Reference Scale M5 Reference Scale M6 Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 3,62 3,68 3,69 3,63 3,86 3,07 3,65 2,25 3,83 4,33 2,36 2,91 3,49 4,59 6,34 7,72 8,26 2,40 8,43 5,71 2,48 8,03 0,80 0,95 1,24 1,75 2,00 2,69 0,66 3,74 1,49 0,57 3,41 Reference Scale M1 Reference Scale M2 Reference Scale M3 Reference Scale M4 Reference Scale M5 Reference Scale M6 Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 3,60 3,60 3,68 3,79 3,78 3,15 3,68 2,34 4,09 4,25 2,42 2,79 3,33 4,70 6,60 7,53 8,16 2,44 8,30 6,56 2,44 7,59 0,78 0,92 1,28 1,74 1,99 2,59 0,66 3,55 1,61 0,58 3,14 0,79 0,94 1,26 1,74 1,99 2,64 0,66 3,65 1,55 0,57 3,27 Wild Type Mutant > 78% Mutant > 12.5% Wild Type Mutant > 78% Mean ratio % V617F S < M1 Wild Type or undetectable M1 S < M2 2-5% M2 S < M % M3 S < M % M4 S < M % M5 S < M % S > M % Mean Ratio Sample equal or superior to Mean Ratio of Reference Scale M1 Mutant Mean Ratio Sample inferior to Mean Ratio Reference Scale M1 undetectable or Wild Type First Edition. Page 22 of 28 DMK03-12

23 In the examples above: For Samples n 1 and 4: Mean ratio Mean ratio M1 Wild Type Samples n 3: Mean ratio Mean ratio M1 Mutant Ratio M3 >Ratio S3 > Ratio M4 % of mutated allele is > 12.5% and < 31% Samples n 5: Mean ratio Mean ratio M1 Mutant Ratio M3 >Ratio S3 > Ratio M4 % of mutated allele is > 78% 5.7. Expression of the results Graphical representation of fluorescence data for Positive and Negative Control; Reference Scale; H2O and Unknown Samples all tested in duplicate PC Rn Fluorescence FAM 530 nm (VF) H2O M6-78% M5-50% M4-31% M3-12% M2-5% M1-2% NC PC H2O 2% 5% 12% 31% 50% 78% Positives Samples Negatives Sam ples NC Rn Fluorescence VIC 560 nm (WT) First Edition. Page 23 of 28 DMK03-12

24 6. Notice This product is intended to be used for life science research only. It is not intended for diagnostic use. IPSOGEN products may not be resold, modified for resale or used to manufacture commercial products without written approval of IPSOGEN. This product is optimised for use in the Polymerase Chain Reaction ("PCR") covered by patents owned by Hoffmann-La Roche, Inc., and F. Hoffmann-La Roche, Ltd. ("Roche"). No license under these patents to use the PCR process is conveyed expressly or by implication to the user by the purchase of this product. The purchase of this product does not convey any right for its use in clinical diagnostic applications. No rights for TaqMan technology under U.S. Patents 5,210,015, 5,487,972 and 5,538,848 or their foreign counterparts, are conveyed by implication to the user by the purchase of this product. Information in this document is subject to change without notice. IPSOGEN assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall IPSOGEN be liable for incidental, special, multiple, or consequential damages in connection with, or arising from the use of this document. JAK2 V617F mutation and uses thereof are protected by international patent application W , and their foreign counterparts, licensed exclusively worldwide to Ipsogen MutaScreen TM is a trademark of IPSOGEN LightCycler is a trademark of Roche Diagnostics, TaqMan is a trademark of Roche Molecular Systems, ABI PRISM and Applied Biosystems are trademarks of Applera Corporation, FAM TM and VIC are trademarks of Applera Corporation 7. Contact information Produced by: IPSOGEN Global Headquarters Luminy Biotech Entreprises Case , avenue de Luminy MARSEILLE cedex 9. France Tel: +33 (0) Fax: +33 (0) support@ipsogen.com infos@ipsogen.com Web: North America - Oncodiagnostics Ipsogen, Inc 83, Maple Avenue. Windsor Connecticut USA Tel: Fax: support@ipsogen.com infos@ipsogen.com Web: MutaScreen TM Kit Reference Scale-V01 Copyright 2007, IPSOGEN Copyright 2007, IPSOGEN. All rights reserved. First Edition. Page 24 of 28 DMK03-12

25 8. Appendix 8.1. ABI Prism 7700 Instrument setting. ABI PRISM 7700 instruments must be running SDS software version 1.7 or later to analyze data from allelic discrimination assays using custom TaqMan MGB probes Preparing a New Plate Read File To prepare a new plate read file: 1 Launch SDS software. 2 Close the untitled window that appears. 3 Create a new plate read file with the following settings: Plate Type Allelic Discrimination Plate Format Standard Plate Run Plate Read 4 Using the Sample Type Setup dialog box, set up sample types: a. Using the Reporter pop-up menu, select the reporter dye attached to the Allele 1 (AL1) probe FAM TM. b. Using the Reporter pop-up menu, select the reporter dye attached to the Allele 2 (AL2) probe VIC. c. Caution: Uncheck the box next to Quencher. TaqMan MGB probes contain a nonfluorescent quencher. d. Click OK. The dialog box closes, and the plate read window becomes active. 5 Label the wells with the sample types. _ Label wells serving as No Template Controls as NTC. _ Label wells serving as Allele 1 controls as AL1. _ Label wells serving as Allele 2 controls as AL2. _ Label wells containing Unknown samples as UNKN. 6 Save the changes to the plate read file Running a PlateRead To run a plate read: 1 Load the reaction plate. a. Place the reaction plate into the sample compartment. b. Pull the heated cover forward. c. Turn the knob clockwise to lower and secure the heated cover over the plate. 2 From the Setup view of a plate read file, click the Show Analysis button. 3 Click the Post-PCR Read button. The instrument will conduct a plate read, which should take about 10 seconds. 4 After the plate read is complete, save the plate read file. 5 Remove the reaction plate from the instrument. IMPORTANT To avoid PCR contamination with amplified product, do not remove the caps from the plate. 6 Discard the reaction plate after analyzing the plate read, when you are confident that the plate read was successful. First Edition. Page 25 of 28 DMK03-12

26 Plate Read Analysis To set up the plate read analysis: 1 Launch SDS software. 2 Close the untitled window that appears. 3 Open the plate read file. a. From the File menu, select Open Plate. b. Choose a plate read file to analyze. c. Click Open. The file appears in the Setup view. 4 Click the Show Analysis button. The Analysis view appears. 5 From the Instrument menu, scroll to Diagnostics, and choose Advanced Options. The Advanced Options dialog box appears. 6 Confirm that the Use Spectral Compensation for Endpoint check box is checked. 7 Click OK. The software displays a warning message requesting you to quit and relaunch the application. 8 Click OK. Note: It is not necessary to quit and relaunch the SDS software at this time Analyzing a Plate Read To analyze a plate read: 1 From the Analysis menu, select Analyze. An event log may appear. If the event log appears, close it. 2 From the Analysis menu, select Allelic Discrimination. The Allelic Discrimination window appears. First Edition. Page 26 of 28 DMK03-12

27 If the Allelic Discrimination window shows autocalls the results require no further modification. If the Allelic Discrimination window does not shows autocalls select Dye Components from the Dye pop-up menu and continue to the next step. 3 Zoom out until all crossmarks are visible in the graph. a. Select the magnifying glass tool by clicking it. b. Click the magnifying glass on the graph to zoom out. Note Select the marks with the lasso tool to confirm that all appropriate wells are selected. 4 Crop and zoom the crossmarks. a. Select the + magnifying glass tool by clicking it. b. Click and drag the + magnifying glass on the graph to crop and magnify all marks. First Edition. Page 27 of 28 DMK03-12

28 5 Repeat step 4 until the marks are clearly clustered in distinct regions of the graph. 6 Manually call the allele types. a. Select the lasso tool by clicking it. b. Circle a cluster of marks with the lasso tool. The software highlights the corresponding wells in the Tray section of the Allelic Discrimination window. 7 Using the Call pop-up menu, designate the appropriate allele type for the selected cluster, based on the location of the graph. _ The software updates the symbols on the graph to match the Legend. _ The software updates the wells in the Tray section of the Allelic Discrimination window with the call. 8 Repeat step 6 and step 7 until you make all calls. 9. Export results. Select File > Export, then click results Cluster Variations The clustering of crossmarks can vary along the horizontal axis (AL1), vertical axis (AL2), or diagonal (AL1/AL2). This variation is due to differences in the extent of PCR amplification, which could result from differences in initial DNA concentration. The example below shows variation in clustering due to differences in the extent of PCR amplification. First Edition. Page 28 of 28 DMK03-12