PointMan™ JAK2 DNA Enrichment Kit
Contents
1. The sample is WT The sample has failed Either there was insufficient amplifiable or PCR inhibitors have prevented a successful test NO Amplicon produced NO Amplicon produced Mutation Details Cosmic IDs are taken from the Catalogue of Somatic Mutations in Cancer www sanger ac uk genetics CGP cosmic Table 2 List of JAK2 mutations and COSMIC IDs Amino Acid Sequence Base Change Cosmic ID Change WT 3 GGA GTA TGT GTC TGT GGA GAC 5 V617F 3 GGA GTA tae Trc TGT GGA GAC5 1848 1849 TG gt CT 25834 V6171 3 GGA GTA TGTJATC TGT GGA GAC5 1849 G gt A 29117 V617F 3 GGA GTA TGTITrc TGTGGAGACS5 1849 G gt T 12600 V617 C618 gt FR 3 GGA GTA TGT TTC GGT GGA GAC 5 1849_1852 GTCT gt TTCC 29281 V617 C618 gt FR 3 GGAGTATGT TIT CGT GGAGAC5 1849_1852 GTCT gt TTTC 27624 V617V 3 GGA GTA TGT G T IGT GGA GAC 5 1851 C gt T 51409 Numerous mutations and deletions in JAK2 codons 617 AND 618 are reported For a full list of the possible mutations enriched by this component of the kit please visit www ekfmolecular com JAK2 User Manual EN 11 13 Page 17 Section 15 Notices and Disclaimers These products are sold exclusively for research and development R amp D use by the purchaser PointMan enrichment kits may not be used for human or veterinary in vitro diagnostic IVD applications and they may not be re sold distributed or re packaged without express written authorisation from EKF Molecular Diagnostics Lice2. in the case of a defect that is apparent on normal visual inspection within five business days of delivery and in the case of a latent defect within a reasonable time of the latent defect having become apparent If you reject a product described as above you may either require us to replace the rejected product or require us to repay the price of the rejected goods in full following return of the goods Section 5 Quality Control As part of our routine quality assurance program all EKF Molecular Diagnostics products are manufactured to ISO9001 standards and monitored to ensure the highest levels of performance and reliability JAK2 User Manual EN 11 13 Page 5 Section 6 Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles All chemicals and biological material must be considered as potentially hazardous Specimens are potentially infectious and must be treated accordingly Discard sample and assay waste in accordance with your local safety regulations Section 7 Introduction to PointMan There is an unmet clinical need for the accurate detection and genoptying of rare somatic mutations The PointMan system is a novel method for selective amplification of genotype specific sequences Unlike allele specific priming PointMan does not in the first instance achieve results by attempting to selectively prime and amplify mutant sequences Such methods are leaky
3. EKF MOLECULAR DIAGNOSTICS PointMan JAK2 DNA Enrichment Kit Instructions for use of PointMan JAK2 DNA enrichment kit PE i MOLECULAR JAK2 User Manual EN 11 13 DIAGNOSTICS Copyright 2013 EKF Molecular iil Diagnostics Ltd No part of this publication may be Avon House reproduced transmitted transcribed stored 19 Stanwell Road in any retrieval system or translated into any Cardiff human or computer language by any means CF64 2EZ or in any form without the prior written United Kingdom permission of EKF Molecular Diagnostics Ltd T 44 0 2920 710 570 Part Number 7137 9006 007 1 F 44 0 2920 710 515 PointMan JAK2 User Manual EU EN Date September 2013 info ekfmolecular com Issue IFUO1 EU EN 04 13 www ekfmolecular com Printed in UK Page 2 JAK2 User Manual EN 11 13 Section 1 Section 2 Section 3 Section 4 Section 5 Section 6 Section 7 Section 8 Section 9 Section 10 Section 11 Section 12 Section 13 Section 14 Section 15 Section 16 Kit Contents Shipping and Storage Product Use Limitations Product Warranty and Guarantee Quality Control Safety Information Introduction to PointMan Reagents and Equipment to Be Supplied by User Sample Material Principle of the Test Bench side Protocol Amplification Protocol Interpretation of Results Data Interpretation Notices and Disclaimers Trademarks JAK2 User Manual EN 11 13 Page 3 Section 1 Kit Contents Kit Contents PointMan JAK2 Enrichmen
4. PointMan enrichment kit purchasers undertake that they are purchasing for the Permitted Use only and that purchasers will not use products for any other use including without limitation diagnostics for medical purposes or commercial purposes and will not resell any products Products are covered by patent applications owned by or licenced to EKF Molecular Diagnostics please see www ekfmolecular com By purchasing any PointMan enrichment kit purchasers acquire a non exclusive licence to use the product for the Permitted Use only EKF Molecular Diagnostics do not warrant that any use of a product will not infringe any patent or any other intellectual property rights whatsoever of any third party Polymerase Chain Reaction PCR is covered by several patents owned by Hoffman Roche Inc and Hoffman LaRoche Ltd Purchase of EKF Molecular Diagnostics kits does not include or provide any licence with respect to any patents owned by Hoffman La Roche or others Section 4 Product Warranty and Guarantee We warrant to you that any product purchased from us will on delivery conform in all material respects to its specification be of satisfactory quality within the meaning of the Sale of Goods Act 1979 as amended and fit for the purpose held out by us be free from material defects in design material and workmanship and comply with all applicable statutory and regulatory requirements You may reject any product which does not comply with the above
5. ation consult the appropriate material safety data sheets MSDSs available from the product supplier DNA sample see below Dedicated pipettes adjustable for PCR master mix preparation Dedicated pipettes adjustable for dispensing of DNA sample Sterile pipette tips with filters Benchtop centrifuge with rotor for 1 5 ml reaction tubes Real time PCR instrument Sterile microcentrifuge tubes for preparing master mixes Section 9 Suitable Sample Material PointMan will work with any high quality source of extracted human DNA and is therefore compatible with a wide range of extraction protocols DNA samples should be quality checked and quantified using a UV spectrophotometer or similar approach Page 10 JAK2 User Manual EN 11 13 General precautions The user should always pay attention to the following Use sterile pipette tips with filters and make sure that pipettes have been calibrated according to the manufacturer s instructions Store and extract positive materials samples and positive controls separately from all other reagents and add them to the reaction mix in a spatially separated facility or within a laminar flow cabinet Thaw all components thoroughly on ice before starting an assay When thawed mix the components by inverting each tube 10 times and centrifuge briefly Do not vortex the mastermix as this may inactivate the Taq DNA polymerase Use extreme caution to prevent contamination of PCRs with
6. er 110 ul 1 positive control 100 ul 3 2 pmol per ul when resuspended Contamination risk contains high copy number of positive control template JAK2 User Manual EN 11 13 Page 13 Preparation of a PointMan reaction Please note that the PointMan JAK2 enrichment kit contains 3 separate primer mixes and control primers Please read tube contents carefully before preparing individual reactions 1 Make up reaction mixes For each sample make up a reaction mix for both the PointMan test and the control well Remember to include additional reactions for the positive and negative controls COMPONENT 1 REACTION PointMan Mastermix with SYBRgreen GREEN 10 ul 2 Pipette 15 ul of relevant reaction mix into each well according to your PointMan experimental plate set up e g SAMPLE SAMPLE SAMPLE SAMPLE SAMPLE POSITIVE NTC 1 2 3 4 5 CTRL e L LI o Ld amp Control primer L J e o L amp 3 Add 5 pl of DNA sample to each reaction Ideally 1 10 ng of high quality DNA should be used For no template controls NTC negative control wells replace sample with RNAse DNAse free water For the 1 positive control well use 5 pl of control template Red Page 14 JAK2 User Manual EN 11 13 Section 12 Amplification Protocol PointMan uses a carefully optimised 4 step cycling parameter Please follow this protocol exactly using a block based real time PCR machine COMPONENT STEP TIME TEMP SSS vn an a a P
7. need extensive optimisation and have limited resolution By contrast PointMan uses allele specific primers for priming on the wild type WT sequence and this then drives those WT templates into terminally extended products that are not available for exponential amplification The variant sequences are uninhibited and therefore free to undergo exponential amplification and enrichment The system uses 4 primers in total fig 1 The internal primers confer the selective power of the assay and are called the enriching primers The external primers are unmodified and are simply used to PCR amplify across the region of interest amplifying primers The enriching primers have the 3 terminal residue situated over the SNP and this residue is homologous with the WT sequence A second important feature of the enriching primers is that they also contain a PCR blocking residue Whist this does not prevent binding of the primer and extension on the genomic DNA template it does prevent the enriching primers from participating in the second and all subsequent rounds of PCR amplification fig 1 The single stranded template made by the enriching primer on the WT sequences blocks the extension of the full length amplicon from the amplifying primer In this way exponential amplification on WT sequences is terminated Page 6 JAK2 User Manual EN 11 13 Section 7 Introduction to PointMan On variant sequences the enriching primer does not extend as it i
8. nse statement Enzymes designed and sold for use in Amplification Patent Rights and or Sequencing Patent Rights A license under US Patents 4 683 202 4 683 195 4 965 188 and 5 075 216 or their foreign counterparts owned by Hoffmann La Roche Inc and F Hoffmann La Roche Ltd Roche has an up front fee component and a running royalty component The purchase price of this product includes limited nontransferable rights under the running royalty component to use only this amount of the product to practice the Polymerase Chain Reaction PCR and related processes described in said patents solely for the research and development activities of the purchaser when this product is used in conjunction with a thermal cycler whose use is covered by the up front fee component Rights to the up front fee component must be obtained by the end user in order to have a complete license to use this product in the PCR process These rights under the up front fee component may be purchased from Perkin Elmer or obtained by purchasing an authorized Thermal Cycler No right to perform or offer commercial services of any kind using PCR including without limitation reporting the results of purchasers activities for a fee or other commercial consideration is hereby granted by implication or estoppel Further information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at The Perkin Elmer Corporation 850 Linc
9. oln Center Drive Foster City California 94404 or at Roche Molecular Systems Inc 1145 Atlantic Avenue Alameda California 94501 The purchase of EKF Molecular Diagnostics reagents cannot be construed as an authorization or implicit license to practice PCR under any patents held by Hoffmann LaRoche Inc or others PointMan Mastermix containing GoTaq Hot Start Polymerase manufactured by Promega Corporation for distribution by EKF Molecular Diagnostics Licensed to Promega under U S Patent Nos 5 338 671 and 5 587 287 and their corresponding foreign patents Page 18 JAK2 User Manual EN 11 13 JAK2 User Manual EN 11 13 Page 19
10. rimer Annealing 20 s 50 CYCLES Enriching Primer Displacement MELT CURVE 60 90 C in 1 C intervals O a Flourogenic data should be collected during these steps through the SYBRgreen FAM channel JAK2 User Manual EN 11 13 Page 15 Section 13 Interpretation of Results Real time PCR traces Analysis of the real time PCR data should give amplification plots with CT values of between 25 and 40 Please note that variant traces will appear before the WT traces due to exponential amplification of the variant sequence 1400 __ 1200 VARIANT gt d 1000 800 600 Melting curve analysis Melting temperatures for PCR products for JAK2 are shown below The melting curve should give a single peak with a melting temperature of 78 C as shown above 350 300 250 200 4 T I gt 150 100 50 L 1 1 45 50 55 60 65 70 75 80 85 90 95 Temperature Celsius Page 16 JAK2 User Manual EN 11 13 Section 14 Table 1 Data Interpretation POINTMAN CONTROL PRIMERS INTERPRETATION PRIMER MIX Armplicen praduced Amplicon produced with Positive for a variant sequence p p melting Tm 78 C Confirm by sequence analysis Reaction successful but no Amplicon produced NO Amplicon produced variant sequences amplified
11. rimers containing blocking moiety 1 complimentary to the wild type sequence bind with high avidity to sample DNA and extend Amplifying primers prime at sites flanking the sequence of interest but are blocked from extension by the high avidity of the extended enriching primers X Variant Enriching primers do not efficiently anneal and extend due to mismatch at the 3 end and stringency of PCR conditions Amplifying primers therefore extend right through the region of interest without hindrance Steps 3 amp 4 Wild Type In the subsequent cycle only products from the enriching primers are available for further replication This replication is terminated by the blocking moieties X and the product does not contain any further priming sites Variant Products of amplifying primers are available for priming and extension Step 5 Wild Type Lack of priming sites for either set of primers ensures only linear replication of starting template Variant Exponential amplification of PCR products is possible Note that there is no risk of false positives from mispriming no extraneous source of variant sequence is introduced in the form of primer dimers unlike allele specific PCR and its variants JAK2 User Manual EN 11 13 Page 9 Section 8 Equipment and Reagents to be Supplied by User When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more inform
12. s the monitoring of product accumulation in real time and also permits melting curve analysis to ensure that a single amplicon corresponding to JAK2 has been amplified Analysis of the SYBRgreen data following PointMan enrichment enables the user to validate that PointManTM has produced a PCR product prior to sample sequencing The PointMan Mastermix cannot be substituted for another enzyme and buffer system as this will greatly affect the sensitivity of the enrichment Page 12 JAK2 User Manual EN 11 13 PointMan Sequencing Primers Yellow To confirm the presence of variant sequences following PointMan enrichment samples can be sequenced using the sequence primers provided to prime the reaction The sequencing primer is unlabelled and is supplied lyophilized Section 11 Bench side Protocol To minimise the risk of contamination with foreign DNA EKF Molecular Diagnostics recommend that all pipetting be performed in a PCR clean environment Ideally this would be a designated PCR cabinet Filter tips are recommended for all pipetting steps 1 Pulse spin each tube in a centrifuge before opening This ensures all products are situated in the base of the tube and prevents reagent loss upon opening the tube 2 Resuspend all components according to the table below using RNase DNase free water supplied COMPONENT VOLUME PointMan primer mix 72 ul Control primer mix 72 ul Forward sequencing primer 110 ul Reverse sequencing prim
13. s displaced by the enzyme during the extension step This allows for full exponential amplification across the variant region fig 1 The PointMan JAK2 kit will exponentially amplify the following sequences if present in the DNA sample For a full list of the mutations enriched please visit www ekfmolecular com WT 3 GGA GTA TGT GTC TGT GGA GAC 35 V617F 3 GGA GTA TGC TTC TGT GGA GAC 5 V6171 3 GGA GTA TGTIATC TGT GGA GAC 5 V617F 3 GGA GTA TGTITTC TGT GGA GAC 5 V617 C618 gt FR 3 GGA GTA TGT TTC CGT GGA GAC 5 V617 C618 gt FR 3 GGA GTA TGTITTT CET GGA GAC 5 V617V 3 GGA GTA TGT GT T TGT GGA GAC 5 1848_1849 TG gt CT 1849 G gt A 1849 G gt T 1849_1852 GTCT gt TTCC 1849_1852 GTCT gt TTTC 1851 C gt T JAK2 User Manual EN 11 13 Page 7 WILD TYPE VARIANT a os nn i ILL n e Oo x e NL e i e 7 o e eom 0 ee E H rn mm o eo d gt x Hi SS 06 e Se a 7 mm o OS 9X A oeo 1 3 3 SS Hi e ul 5 e ng m mmmmums 09 9 c 552525255 Figure 1 PointMan amplifies the SNP sequence but blocks amplification of the WT sequence Page 8 JAK2 User Manual EN 11 13 Step 1 amp 2 Wild Type Enriching p
14. synthetic control material We recommend using separate dedicated pipettes for setting up reaction mixes and adding DNA template Preparation and dispensing of reaction mixes must be carried out in a separate area to the addition of sample Reagents for the PointMan JAK2 kit have been optimally diluted EKF Molecular Diagnostics do not recommend further dilution of reagents as this may result in a loss of performance We do not recommend using reaction volumes of less than 15 ul as this may increase the risk of false negatives All reagents in the PointMan JAK2 Kit are formulated specifically for use with the stated tests All reagents supplied in the PointMan JAK2 Kit are intended to be used solely with the other reagents in the same Kit Substitutions to the reagents in the kit must not be made if optimal performance is to be maintained Only use the Taq DNA polymerase that is provided in the kit Do not substitute with Tag DNA polymerase from other kits of the same or any other type or with Taq DNA polymerase from another supplier Ensure that all instruments have been checked and calibrated according to the manufacturer s recommendations JAK2 User Manual EN 11 13 Page 11 Section 10 Principles of the Test PointMan Primer Mix Black This primer mix contains the 4 primers required for a PointMan reaction When used in conjunction with the PointMan Mastermix and user supplied DNA sample this reaction will selectively enrich
15. t Kit The PointMan JAK2 kit contains 3 separate primer mixes and control primers Please read tube contents carefully before preparing individual reactions e PointMan JAK2 primer mix 24 reactions BLACK Control primer mix JAK2 24 reactions WHITE e PointMan JAK2 Mastermix with SYBRgreen 75 reactions GREEN e Sequencing primer forward JAK2 100 reactions BLUE e Sequencing primer reverse JAK2 100 reactions BLUE e 1 Positive control JAK2 20 reactions RED e RNase DNase free water WHITE Section 2 Shipping and Storage The PointMan JAK2 kit is shipped frozen and must still be frozen upon arrival If the PointMan JAK2 kit is not frozen upon arrival the outer packing has been opened during transit the shipment does not contain a packing note instruction booklet or the reagents please contact the EKF Molecular technical services department info ekfmolecular com The PointMan JAK2 kit should be stored at 15 C to 25 C and protected from sunlight When stored under the recommended storage conditions in the original packaging the PointMan JAK2 kit is stable for 6 months from the date of purchase Repeated thawing and freezing should be avoided We recommend a maximum of 7 freeze thaw cycles Page 4 JAK2 User Manual EN 11 13 Section 3 Product Use Limitations EKF Molecular Diagnostics sell products solely for use in laboratory research to gain information for use by the purchaser the Permitted Use By purchasing a
16. the PCR reaction for variant sequences if present Following PointMan reaction completion the enriched product can be sequenced to confirm if the variant sequence is present in the tissue sample Control Primer Mix White TThe PointMan reaction blocks amplification of the wide type WT sequence thus it follows that clinical samples which contain only the WT sequence may not produce any amplification product Experimentally it has been demonstrated that if the sample contains less than 1000 copies of WT sequence no PCR product will accumulate For this reason it is essential to confirm that the sample has been extracted correctly and is competent to support PointMan PCR amplification The control primer mix contains only the amplifying primers without the enriching primers This mix will therefore produce a post reaction product on both WT and variant sequences The control primer mix is used to confirm that a biological sample is competent for PCR and therefore valid for PointMan analysis A positive signal from the control assay primers is essential to enable validation of negative results PointMan Mastermix with SYBRgreen Green The PointMan Mastermix is specifically formulated to provide optimal blocking of amplification on WT sequences whist allowing efficient amplification of variant sequences Consistent with the PointMan process outlined above the enzyme is exonuclease deficient The mastermix also contains SYBRgreen This allow
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