DNA Enrichment Kit
Contents
1. Step 1 amp 2 Wild Type Enriching primers containing blocking moiety X complementary to the wild type sequence bind with high avidity to sample DNA Step 1 Enriching primers with blocking moiety X complimentary to the wild type sequence and a mismatch at the 3 end to the variant mutant sequence the base of interest is shown as wild type blue oval 6 variant red oval amp Amplifying primers complimentary to sequences flanking the region of interest Step 2 Wild Type Enriching primers anneal to wild type with high avidity due to match at 3 end Extension from enriching primers occurs further increasing avidity Amplifying primers anneal and extend but extension is terminated by presence of the extending enriching primer 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 the product of extension from the enriching primers provides priming sites for further replication but extension is terminated by the blocking moieties and therefore the product does not itself contain any priming sites Therefore no further replication is possible in subsequent cycles and the reaction is driven into linear amplification based only on the original sample material Variant I2. 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 On variant sequences the enriching primer does not extend as it is displaced by the enzyme during the extension step This allows for full exponential amplification across the variant region fig 1 The PointMan KRAS kit will exponentially amplify deletions and alteration in the sequences highlighted in red below For a full list of the mutations enriched please visit www ekfmolecular com Codon 12 3 GTT GGA GCT GGT GGC GTA GGC AAG AGT 5 Codon 13 3 GTT GGA GCT GGT GGC GTA GGC AAG AGT 5 Codon 61 3 GAC ACA GCA GGT CAA GAG GAG TAC AGT 5 KRAS User Manual EN 11 13 Page 7 WILD TYPE VARIANT os nn i ILL un e Oo x e _ NL 0 e i e in x p L6 ee z Tr d D eo gt x Hi 06 e Se a 7 mm o OS OX A oeo 1 o 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 KRAS User Manual EN 11 13
3. 3 4 5 CTRL e e E e Control primer amp J amp e 3 Add 5 ul 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 ul of control template Page 14 KRAS 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 SS MS an a a Primer Annealing 20s 50 CYCLES Enriching Primer Displacement MELT CURVE 60 90 C in1 C intervals OO ae Flourogenic data should be collected during these steps through the SYBRgreen FAM channel KRAS 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 in the region of CT 25 40 Please note that variant traces for codon 12 13 will appear before the WT traces due to exponential replication of variant sequence 1 750 000 1 500 000 1 250 000 VARIANT gt 1 000 000 ARn 750 000 500 000 WT 250 000 o 250 000 2 4 6 2 M 6 Wn Z2 2 A2 5 2 D 2 amp 4 s a 9 Cycle Melting curve analysis The melting curve for codon 12 13 should give a single peak with a melting temperature of
4. 61 are reported the most common are listed below For a full list of the possible mutations enriched by this component of the kit please visit www ekfmolecular com Cosmic IDs are taken from the catalogue of Somatic Mutations in Cancer www sanger ac uk genetics CGP cosmic Page 18 KRAS User Manual EN 11 13 Section 14 Table 1 Data Interpretation Table 2 List of KRAS mutations and COSMIC IDs Mutation Gly12Asp Gly12Ala Gly12Val Gly12Ser Gly12Arg Gly12Cys Gly13Asp Gin 1Lys Gin 1Lys Gin 1Lys GIn61Glu Gin 1Arg Gin 1Lys Gin 1Arg GIn61Pro Gln61His Gln61His Sequence S GTAGTTGGAGCTGATGGCGTAGGCAAGAGTS S GTAGTTGGAGCTGCTGGCGTAGGCAAGAGTS S GTAGTTGGAGCTGTTGGCGTAGGCAAGAGTS 3 GTAGTTGGAGCTAGTGGCGTAGGCAAGAGTS 3 GTAGTTGGAGCTCGTGGCGTAGGCAAGAGTS 3 GTAGTTGGAGCTTGTGGCGTAGGCAAGAGTS S GTAGTTGGAGCTGGTGACGTAGGCAAGAGTS 3 GAC ACA GCA GGA AAA GAG GAG TAC agt5 3 GAC ACA GCA GGC AAA GAG GAG TAC AGTS 3 GAC ACA GCA GGT AAA GAG GAG TAC AGTS 3 GAC ACA GCA GGT GAA GAG GAG TAC AGTS 3 GAC ACA GCA GGT CGT GAG GAG TAC AGT5 3 GAC ACA GCA GGT CTA GAG GAG TAC AGTS 3 GAC ACA GCA GGT CGA GAG GAG TAC AGT5 3 GAC ACA GCA GGT CCA GAG GAG TAC AGTS 3 GAC ACA GCA GGT CAC GAG GAG TAC AGTS 3 GAC ACA GCA GGT CAT GAG GAG TAC AGTS Base Change 35 G gt A 35 G gt C 35 G gt T 34 G gt A 34 G gt C 34 G gt T 38 G gt A 180_181 TC gt AA 180_181 TC gt CA 181 C gt A 181 C gt G 180_181 AA gt GT 182 A gt T 182 A gt G 182
5. 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 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 KRAS User Manual EN 11 13 Page 5 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 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 genotyping of rare somati
6. 80 C as shown below 600000 0 500000 0 400000 0 300000 0 Derivative Reporter Rn 200000 0 100000 0 0000 0 vo 850 ma 10 800 Tm 79 78 Temperature C Page 16 KRAS User Manual EN 11 13 Section 13 Interpretation of Results Amplification Plot 850 000 800 000 1 750 000 700 000 650 000 4 600 000 1 550 000 1 500 000 450 000 lt 400 000 350 000 300 000 250 000 1 200 000 150 000 1 100 000 50 000 1 of 50 000 Melting curve analysis The melting curve for codon 61 should give a single peak with a melting temperature of 80 5 C as shown above Melt Curve 360000 0 300000 0 260000 0 200000 0 150000 0 Derivative Reporter Rn 100000 0 0000 0 00 0 05 0 700 10 900 Tm 90 52 Temperature C 850 90 0 950 KRAS User Manual EN 11 13 Page 17 Section 14 Table 1 Data Interpretation POINTMAN CONTROL PRIMERS INTERPRETATION PRIMERS MIX Aivblicon produced Amplicon produced with Positive for a variant sequence p p melting Tm 80 C Confirm by sequence analysis Amplicon produced NO Amplicon produced Reaction successful but no variant sequences amplified The sample has failed Either there was insufficient amplifiable NO Amplicon produced NO Amplicon produced DNA or PCR inhibitors have prevented a successful test Mutation Details Numerous mutations and deletions in KRAS exons 12 13 and
7. A gt T 183 A gt C 183 A gt T Cosmic ID 521 522 520 517 518 516 532 87298 12729 549 550 1168052 553 552 551 554 555 KRAS User Manual EN 11 13 Page 19 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 authorization from EKF Molecular Diagnostics License 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 produc
8. EKF MOLECULAR DIAGNOSTICS PointMan KRAS codon 12 13 61 DNA Enrichment Kit Instructions for use of PointMan KRAS DNA enrichment kit PE p MOLECULAR KRAS User Manual EN 11 13 DIAGNOSTICS CopyrightO 2013 EKF Molecular wal Diagnostics Ltd No part of this publication may be Kv nklause 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 3012 0212 F 44 0 2920 710 515 PointMan KRAS User Manual EU EN Date September 2013 info ekfmolecular com Issue IFUO1 EU EN 04 13 www ekfmolecular com Printed in UK Page 2 KRAS 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 KRAS User Manual EN 11 13 Page 3 Section 1 Kit Contents Kit Contents P
9. 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 allows the monitoring of product accumulation in real time and also permits melting curve analysis to ensure that a single amplicon corresponding to BRAF has been amplified Analysis of the SYBRgreen data following PointMan enrichment enables the user to validate that PointMan 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 KRAS 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 lyophilised Section 11 Bench side Protocol To minimise the risk of contamination with foreign DNA EKF Molecular recommend that all pipetting be perform
10. c 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 may be error prone 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 Page 6 KRAS User Manual EN 11 13 Section 7 Introduction to PointMan 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 Whilst 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
11. ded We recommend a maximum of 7 freeze thaw cycles Page 4 KRAS 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 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
12. ed 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 TIO ul Reverse sequencing primer TIO ul 1 positive control 100 ul 3 2 pmol per ul when resuspended Contamination risk contains high copy number of positive control template KRAS User Manual EN 11 13 Page 13 Preparation of a PointMan reaction Please note that the PointMan KRAS 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 primer mix BLACK 2 yl PointMan Mastermix with SYBRgreen GREEN 10 ul RNAse DNAse free water CLEAR 3 yl 2 Pipette 15 pl of relevant reaction mix into each well according to your PointMan experimental plate set up e g SAMPLE SAMPLE SAMPLE SAMPLE SAMPLE osmve 1 2
13. librated 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 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 KRAS 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 KRAS Kit are formulated specifically for use with the stated tests All reagents supplied in the PointMan KRAS 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
14. n subsequent cycles stemming from the full replicons unencumbered by blocking moieties exponential amplification proceeds in the usual way Note that there is no risk of false positives from mispriming no extraneous source of the variant sequence is introduced in the form of primers unlike allele specific PCR and its variants KRAS 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 information 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 sample DNA 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 KRAS 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 ca
15. ointMan KRAS Kit The PointMan KRAS kit contains 3 separate primer mixes and control primers Please read tube contents carefully before preparing individual reactions PointMan KRAS 12 13 primer mix 24 reactions BLACK PointMan KRAS 61 primer mix 24 reactions BLACK Control primer mix KRAS 12 13 24 reactions WHITE Control primer mix KRAS 61 24 reactions WHITE PointMan KRAS Mastermix with SYBRgreen 225 reactions GREEN Sequencing primer forward KRAS 12 13 100 reactions YELLOW Sequencing primer reverse KRAS 12 13 100 reactions YELLOW Sequencing primer forward KRAS 61 100 reactions YELLOW Sequencing primer reverse KRAS 61 100 reactions YELLOW 196 Positive control KRAS 12 13 8 reactions RED 196 Positive control KRAS 61 8 reactions RED RNase DNase free water CLEAR Section 2 Shipping and Storage The PointMan KRAS kit is shipped frozen and must still be frozen upon arrival If the PointMan KRAS 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 infoGekfmolecular com The PointMan KRAS 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 KRAS kit is stable for 6 months from the date of purchase Repeated thawing and freezing should be avoi
16. substitute with Taq 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 KRAS User Manual EN 11 13 Page 11 Section 1O 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 the PCR reaction for variant sequences if present Following PointMan reaction completion the enriched product can be sequenced to confirm presence at the variant sequence in the DNA sample Control Primer Mix White The PointMan reaction blocks amplification of the wild 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
17. t 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 Lincoln 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 authorisation or implicit license to practice PCR under any patents held by Hoffmann LaRoche Inc or others PointManTM 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 20 KRAS User Manual EN 11 13 KRAS User Manual EN 11 13 Page 21 Page 22 KRAS User Manual EN 11 13 KRAS User Manual EN 11 13 Page 23
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