KRAS Pyro Handbook
Contents
1. OSHA Occupational Safety and Health Administration United States of America t ACGIH American Conference of Government Industrial Hygienists United States of America COSHH control of Substances Hazardous to Health United Kingdom Be sure to observe federal state and local environmental regulations for the disposal of laboratory waste Important point before starting This protocol requires high purity water Milli Q 18 2 MQ x cm www millipore com or equivalent Procedure B1 Ensure that no vacuum is applied to the vacuum tool Make sure that the vacuum is closed Off and the vacuum pump is switched off B2 Discard any solutions left in the troughs B3 Rinse the troughs with high purity water or replace them if necessary B4 Empty the waste container The cap can be removed without disconnecting the tubing KRAS Pyro Handbook 09 2010 35 B5 If the vacuum workstation must be cleaned for example due to dust or spillage follow the instructions in the PyroMark Q24 User Manual References QIAGEN maintains a large up to date online database of scientific publications utilizing QIAGEN products Comprehensive search options allow you to find the articles you need either by a simple keyword search or by specifying the application research area title etc For a complete list of references visit the QIAGEN Reference Database online at www qiagen com RefDB search asp or contact QIAGEN Technic2. page 25 and Appendix A page 33 8 KRAS Pyro Handbook 09 2010 Workflow of KRAS Pyro procedure Assay and run setup Sample preparation PCR Protocol 2 Assay file setup Appendix A i i Immobilization Protocol 3 Y Run file setup Protocol 1 Preparation of samples Protocol 4 Bc C n PyroMark Q24 run Protocol 5 Y Analysis of PyroMark Q24 run Protocol 6 Y Report KRAS Pyro Handbook 09 2010 9 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 E DNA isolation kit see DNA isolation page 11 Pipets adjustable Sterile pipet tips with filters for PCR setup Benchtop microcentrifuge Thermal cycler and appropriate PCR tubes Streptavidin Sepharose High Performance GE Healthcare cat no 17 5113 01 www gelifesciences com PyroMark Q24 cat no 9001514 PyroMark Q24 Software cat no 9019062 PyroMark Q24 Plate cat no 979201 PyroMark Q24 Cartridge cat no 979202 PyroMark Q24 Vacuum Workstation cat no 9001518 220V or 9001516 110V or 9001519 100V Plate mixer for immobilization to beads Heating block capable of attaining 80 C 24 well PCR plate or strips Strip caps High purity water Milli Q 18 2 MQ x cm or equivalent Note Sufficient water is supplied to diss
3. It is not necessary to keep PCR tubes on ice since HotStarTaq DNA Polymerase is inactive at room temperature 4 Add 5 ul template DNA 2 10 ng of genomic DNA to the individual PCR tubes see Table 3 and mix thoroughly A negative control without template DNA should always be included Include a reaction with unmethylated control DNA as positive control see Controls page 12 Table 3 Preparation of PCR Component Volume reaction Reaction mix 20 ul Sample DNA 5 ul Total volume 25 pl SSS Se 16 KRAS Pyro Handbook 09 2010 5 Program the thermal cycler according to the manufacturer s instructions using the conditions outlined in Table 4 Table 4 Optimized cycling protocol Comments Initial activation 15 min 95 C HotStarTaq DNA step Polymerase is activated by this heating step 3 step cycling Denaturation 20s 95 C Annealing 30s 53 C Extension 20s 72 6 Number of cycles 42 Final extension 5 min 22 0 6 Place the PCR tubes in the thermal cycler and start the cycling program 7 After amplification proceed with Protocol 3 Immobilization of PCR Products to Streptavidin Sepharose High Performance Beads page 18 KRAS Pyro Handbook 09 2010 17 Protocol 3 Immobilization of PCR Products to Streptavidin Sepharose High Performance Beads This protocol is for immobilization of template DNA to Streptavidin Sepharose High Performance GE Healthcare prior to analysis on the
4. Insufficient enzyme or Make sure to fill the PyroMark Q24 Cartridge substrate mix for all according to the Pre Run Information in the wells Tools menu b Reagents incorrectly Prepare the PyroMark Q24 Gold Reagents stored or diluted according to the instructions supplied with the reagents 32 KRAS Pyro Handbook 09 2010 Appendix A Setting Up KRAS Pyro Assays If the KRAS Plug in Report has been installed use the assay setups supplied for codons 12 and 13 and codon 61 The following steps do not need to be performed The KRAS Plug in Report can be obtained from pyro plugin Qgiagen com We strongly recommend the use of the KRAS Plug in Report over manual analysis After installation of the plug in or each time new software is installed or upgraded on the office computer the correct function of the Plug in should be verified as described in the KRAS Plug In Quick Guide If the KRAS Plug in Report has not been installed the assay file must be set up manually before running the KRAS Pyro assay for the first time as described below Procedure KRAS codons 12 and 13 A1 Set up the assay for KRAS codons 12 position 2 and 13 position 2 by using the PyroMark Q24 Software A2 Click in the toolbar and select New AQ Assay A3 Type the following sequence in Sequence to Analyze GNTGRCGTAGGC The most frequent mutations in codon 12 will be detected in nucleotide 35 second position using this sequence to Analyze To ana
5. PyroMark Q24 System Things to do before starting amp Allow all required reagents and solutions to reach room temperature 15 25 C before starting Procedure 1 Gently shake the bottle containing Streptavidin Sepharose High Performance until it is a homogeneous solution 2 Prepare a master mix for DNA immobilization according to Table 5 Prepare a volume 1096 greater than that required for the total number of reactions to be performed Table 5 Master mix for DNA immobilization Component Volume sample Streptavidin Sepharose High Performance 2 ul PyroMark Binding Buffer 40 ul Water supplied 28 ul Total volume 70 pl 3 Add 70 ul of the master mix to wells of a 24 well PCR plate or strips as predefined in the run setup see Protocol 1 Run Setup for the PyroMark Q24 System page 13 4 Add 10 pl biotinylated PCR product from Protocol 2 to each well containing master mix as predefined in the run setup see Protocol 1 Run Setup for the PyroMark Q24 System page 13 The total volume per well should be 80 ul after addition of the master mix and PCR product 5 Seal the PCR plate or strips using strip caps Ensure that no leakage is possible between the wells 18 KRAS Pyro Handbook 09 2010 6 Agitate the PCR plate at room temperature 15 25 C for 5 10 min at 1400 rpm During this step prepare the PyroMark Q24 Vacuum Workstation for sample preparation as described in the PyroMark Q24 User Manua
6. To All when the Apply Analysis Setup window appears To reanalyze and target mutations at nucleotide 182 second position of Codon 61 change the Sequence to Analyze of the Codon 61 assay to the following sequence CTCTHGACCTG To reanalyze and target mutations at nucleotide 181 first position of Codon 61 change the Sequence to Analyze of the Codon 61 assay to the following sequence CTCTTSACCTG Note Ensure the threshold for single peak height is set to 30 RLU Rerunning samples for detection of low level mutations It is strongly recommended that a wild type control sample is included in every run for comparison Any sample showing a mutation frequency higher than the corresponding position in the wild type control sample should be examined in relation to the table showing the limit of detection see Table 7 page 28 If using the KRAS Plug in Report this is performed automatically As a guide samples that have a suspected mutation in the range from LOD Table 7 to LOD 3 units should be reanalyzed in duplicate together with a wild type control sample in duplicate If using the Plug in Report step 5 a warning will be issued if this occurs If both duplicates give the same result as KRAS Pyro Handbook 09 2010 27 the original analysis and are visibly different from the wild type control then the sample can be considered to be positive for the mutation In case of a suspected GGT gt GTT mutati
7. 0 Fax 0800 28 10 19 Technical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fax 55 11 5079 4001 Technical 0800 557779 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 86 21 3865 3865 Fax 86 21 3865 3965 Technical 800 988 0325 Denmark Orders 80 885945 Fax 80 885944 Technical 80 885942 Finland Orders 0800 914416 Fax 0800 914415 Technical 0800 914413 France Orders 01 60 920 926 Fax 01 60 920 925 Technical 01 60 920 930 Offers 01 60 920 928 Germany Orders 02103 29 12000 Fax 02103 29 22000 Technical 02103 29 12400 Hong Kong Orders 800 933 965 Fax 800 930 439 Technical 800 930 425 Ireland Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 Italy Orders 800 789 544 Fax 02 334304 826 Technical 800 787980 Japan Telephone 03 6890 7300 Fax 03 5547 0818 Technical 03 6890 7300 Korea South Orders 080 000 7146 Fax 02 2626 5703 Technical 080 000 7145 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 Mexico Orders 01 800 7742 639 Fax 01 800 1122 330 Technical 01 800 7742 436 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859 Fax 800 18817 Technical 800 18712 Singapore Orders 1800 742 4362 Fax 65 6854 8184 Technical 1800 742 4368 Spain Orders 91 630 7
8. 050 Fax 91 630 5145 Technical 91 630 7050 Sweden Orders 020 790282 Fax 020 790582 Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 0000o USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN m Sample amp Assay Technologies
9. Q24 System 23 B 6 Analysis of a PyroMark Q24 Run 25 Troubleshooting Guide 31 Appendix A Setting Up KRAS Pyro Assays 33 Appendix B Emptying the Waste Container and Troughs 35 References 36 Ordering Information 37 KRAS Pyro Handbook 09 2010 3 Kit Contents KRAS Pyro Kit box 1 2 KRAS Pyro Kit 24 Catalog no 970460 Number of reactions 24 Seq Primer KRAS 12 13 24 ul Seq Primer KRAS 61 24 ul PCR Primer KRAS 12 13 24 ul PCR Primer KRAS 61 24 ul PyroMark PCR Master Mix 2x 850 ul CoralLoad Concentrate 10x 1 2 ml H O 3x 1 9 ml Unmethylated Control DNA 10 ng ul 100 ul EEG 4 KRAS Pyro Handbook 09 2010 Buffers and Reagents box 2 2 Buffers and Reagents PyroMark Binding Buffer 10 ml PyroMark Annealing Buffer 10 ml PyroMark Denaturation Solution 250 ml PyroMark Wash Buffer 10x 25 ml Enzyme Mixture 1 vial Substrate Mixture 1 vial dATPaS 1180 ul dCTP 1180 ul dGTP 1180 ul dTTP 1180 ul Handbook 1 Contains sodium hydroxide Shipping and Storage The KRAS Pyro Kit is shipped in two boxes The KRAS Pyro Kit box 1 2 is shipped on dry ice PyroMark PCR Master Mix CoralLoad Concentrate unmethylated control DNA and all primers should be stored at 15 to 25 C upon arrival The Pyro Buffers and Reagents box 2 2 containing buffers enzyme mixture substrate mixture dATPaS dCTP dGTP and dTTP the reagents for Pyrosequencing analysis is shipped on cool packs These component
10. September 2010 KRAS Pyro Handbook For quantitative measurements of mutations in codons 12 13 and 61 of the human KRAS gene QIAGEN Sample amp Assay Technologies QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www giagen com Contents Kit Contents 4 Shipping and Storage 5 Product Use Limitations 6 Technical Assistance 6 Product Warranty and Satisfaction Guarantee 6 Safety Information 7 Quality Control 7 Introduction 8 Principle and procedure 8 Equipment and Reagents to Be Supplied by User 10 Important Notes 11 General precautions 11 Sample material 11 DNA isolation 11 Controls 12 Protocols E 1 Run Setup for the PyroMark Q24 System 13 B 2 PCR Using the PCR Reagents Supplied with the KRAS Pyro Kit 15 H 3 Immobilization of PCR Products to Streptavidin Sepharose High Performance Beads 18 B 4 Preparation of Samples Prior to Pyrosequencing Analysis on the PyroMark Q24 20 B 5 Running the PyroMark
11. TCTTSACCTG Note Ensure the threshold for single peak height is set to 30 RLU A3 Manually add the following Dispensation Order GCTCAGTCAGACT Figure 11 Histogram for codon 61 nucleotide 183 with the Sequence to Analyze CTCDTGACCTG Figure 12 Histogram for codon 61 nucleotide 182 with the Sequence to Analyze CTCTHGACCTG Figure 13 Histogram for codon 61 nucleotide 181 with the Sequence to Analyze CTCTTSACCTG 34 KRAS Pyro Handbook 09 2010 A4 Click the Analysis Parameters tab and increase Peak Height Threshold Required peak height for Passed quality to 30 A5 Click amp in the toolbar and save the assay as KRAScodon 61 Appendix B Emptying the Waste Container and Troughs WARNING Hazardous chemicals A The Denaturation Solution used with the vacuum workstation contains sodium hydroxide which is irritating to eyes and skin Always wear safety glasses gloves and a lab coat The responsible body e g laboratory manager must take the necessary precautions to ensure that the surrounding workplace is safe and that the instrument operators are not exposed to hazardous levels of toxic substances chemical or biological as defined in the applicable Material Safety Data Sheets MSDSs or OSHA ACGIH or COSHH documents Venting for fumes and disposal of wastes must be in accordance with all national state and local health and safety regulations and laws
12. al Services or your local distributor SSS EEE 36 KRAS Pyro Handbook 09 2010 Ordering Information Product Contents Cat no KRAS Pyro Kit 24 For 24 reactions on PyroMark Q24 970460 Systems Seq Primers PCR Primers Unmethylated Control DNA PyroMark PCR Master Mix CoralLoad Concentrate PyroMark Binding Buffer PyroMark Annealing Buffer PyroMark Denaturation Solution PyroMark Wash Buffer Enzyme Mixture Substrate Mixture dATPaS dCTP dGTP dTTP and H O PyroMark Q24 Sequence based detection platform for 9001514 Pyrosequencing of 24 samples in parallel PyroMark Q24 Vacuum Vacuum Workstation for preparing 9001518 Workstation 24 samples in parallel from PCR 220 V product to single stranded template 9001516 110 V 9001519 100 V PyroMark Q24 Analysis software 9019062 Software Accessories PyroMark Q24 Plate 24 well sequencing reaction plate 979201 100 PyroMark Q24 Cartridges for dispensing nucleotides 979202 Cartridge 3 and reagents PyroMark Vacuum Prep Reusable filter probes for PyroMark 979010 Filter Probe 100 Vacuum Workstation Q96 and Q24 PyroMark Control For installation check of system 979203 Oligo PyroMark Q24 For performance confirmation of 979204 Validation Oligo system KRAS Pyro Handbook 09 2010 37 Ordering Information Product Related products QlAamp DNA FFPE Tissue Kit 50 EZ1 DNA Tissue Kit 48 PAXgene Tissue Containers 10 PAXgene Tissue DNA Kit 50 QlAamp DSP DNA Bl
13. amples avoid placing samples with high signal intensities next to no template control wells b PCR contamination Use sterile pipet tips with filters Store and extract materials such as specimens plasmid controls and amplicons separately from PCR reagents Poor or unexpected sequence a Low quality genomic Low quality genomic DNA can cause the PCR to DNA fail Analyze PCR samples using an electrophoretic technique using for example the QlAxcel System or agarose gel electrophoresis b Unexpected rare A Check or Failed quality assessment can be mutation caused by an unexpected pattern of peaks This might indicate an unexpected mutation which is not analyzed by the standard Sequence to Analyze These samples should be analyzed using the alternative Sequence to Analyze considering unexpected mutations Check or failed result a Rare mutation not Adjust the sequence to analyze in the assay setup defined in the assay see Appendix A page 33 and reanalyze the setup run KRAS Pyro Handbook 09 2010 31 Comments and suggestions b Low peak height Handling errors in PCR setup or sample preparation prior to Pyrosequencing can result in low peaks It is recommended to reanalyze the sample High background Incorrect storage of Store nucleotides at 2 8 C Storage at nucleotides 15 to 25 C can cause an increase in the background No signals in positive control Unmethylated Control DNA a
14. cing primer greater than that required for the total number of samples to be sequenced for the number of samples one extra Table 6 Example dilution of the sequencing primers Volume for Component Volume sample 9 1 reactions Seq Primer KRAS 12 13 or 0 8 ul 8 ul Seq Primer KRAS 61 E PyroMark Annealing Buffer 24 2 ul 242 ul Total volume 25 pl 250 ul 20 KRAS Pyro Handbook 09 2010 2 Add 25 ul of diluted sequencing primer to each well of the PyroMark Q24 Plate according to the run setup see Protocol 1 Run Setup for the PyroMark Q24 page 13 Keep one of the PyroMark Q24 Plate Holders supplied with the PyroMark Q24 Vacuum Workstation at room temperature 15 25 C and use it as support when preparing and moving the plate 3 Place the PCR plate or strips from Protocol 3 and the PyroMark Q24 Plate on the worktable Ensure that the plate is in the same orientation as when samples were loaded Figure 2 Placement of PCR plate or strips and PyroMark Q24 plate on the vacuum workstation 4 Apply vacuum to the tool by opening the vacuum switch 5 Carefully lower the filter probes into the PCR plate or strips to capture the beads containing immobilized template Hold the probes in place for 15 s Take care when picking up the tool Sepharose beads sediment quickly If more than 1 min has elapsed since the plate or strips was agitated agitate again for 1 min before capturing th
15. computer s USB port 2 Move the run file from the USB stick to the desired location on the computer using Windows Explorer 3 Open the run file in AQ mode of PyroMark Q24 Software either by selecting Open in the File menu or by double clicking the file 6 in the shortcut browser 4 There are 2 methods for analyzing the run If using the KRAS Plug in Report go to step 5 If using the AQ analysis integral to the PyroMark Q24 System go to step 6 Note We strongly recommend using the KRAS Plug in Report This report ensures that the correct LODs are used and different sequences to analyze are automatically used to detect all mutations 5 Using the KRAS Plug in Report To generate a report select AQ Add On Reports KRAS and Codon 12 and 13 or Codon 61 from Reports in the menu P PyromMark Q24 2 0 6 AQ Run Analysis G _ 100318_KRAS_Primer Test 100318_KRAS_Primertest2 3 File Tools Reports Window Help TL g mL AQ Analysis Statistics AQ Analysis Results AQ Pyrogram Report AQ Full Report AQ Add On Reports gt SNP Analysis Results Codon 12 and 13 Codon 61 SNP Pyrogram Report SNP Full Report SNP Overview Report or LLLI li III Y KRAS Pyro Handbook 09 2010 25 The wells will automatically be analyzed for all mutations for which LOD is given in Table 7 page 28 The results will be presented in an overview table see example below followed by the detailed resul
16. e beads 6 Transfer the tool to the trough containing 40 ml 70 ethanol trough 1 Flush the filter probes for 5 s 7 Transfer the tool to the trough containing 40 ml Denaturation Solution trough 2 Flush the filter probes for 5 s 8 Transfer the tool to the trough containing 50 ml Wash Buffer trough 3 Flush the filter probes for 10 s 9 Raise the tool up and back beyond 90 vertical for 5 s to drain liquid from the filter probes KRAS Pyro Handbook 09 2010 21 Figure 3 Illustration of the vacuum tool raised to beyond 90 vertical 10 While the tool is held over the PyroMark Q24 Plate close the vacuum switch on the tool Off 11 Release the beads in the plate containing the Seq Primers by shaking the tool gently from side to side 12 Transfer the tool to the trough containing high purity water trough 4 and agitate the tool for 10 s 13 Wash the filter probes by lowering the probes into high purity water trough 5 and applying vacuum Flush the probes with 70 ml high purity water 14 Raise the tool up and back beyond 90 vertical for 5 s to drain liquid from the filter probes 15 Close the vacuum switch on the tool Off and place the tool in the Parking P position 16 Turn off the vacuum pump At the end of a working day liquid waste and remaining solutions should be discarded and the PyroMark Q24 Vacuum Workstation should be checked for dust and spillage see Appendix B page 35 17 Heat
17. ecommended for DNA purification from the indicated human sample types for use with the KRAS Pyro Kit Carry out the DNA purification according to the instructions in the kit handbooks KRAS Pyro Handbook 09 2010 11 Table 1 DNA purification kits recommended for use with the KRAS Pyro Kit Catalog number Sample material Nucleic acid isolation kit QIAGEN QIAomp DNA FFPE Tissue Kit 50 56404 EZ1 DNA Tissue Kit 48 953034 s PAXgene Tissue Containers 10 765112 PAXgene Tissue DNA Kit 50 767134 Blood QlAamp DSP DNA Blood Mini Kit 61104 Following the protocol for use with paraffin embedded tissue The EZ1 DNA Tissue Kit should be used in combination with the EZ1 Advanced cat no 9001410 or 9001411 and the EZ1 Advanced DNA Paraffin Section Card cat no 9018298 with the EZ1 Advanced XL cat no 9001492 and the EZ1 Advanced XL DNA Paraffin Section Card cat no 9018700 or with the BioRobot EZ1 cat no 9000705 no longer available and the EZ1 DNA Paraffin Section Card cat no 9015862 Controls Unmethylated control DNA is included in the product as a positive control for PCR and sequencing reactions In addition a negative control without template DNA should always be included E 12 KRAS Pyro Handbook 09 2010 Protocol 1 Run Setup for the PyroMark Q24 System Things to do before starting B I If the KRAS Plug in Report has not been installed create an Assay Setup as described in Appendi
18. esitate to contact us QIAGEN customers are a major source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at QIAGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please see our Technical Support Center at www giagen com Support or call one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www qiagen com Product Warranty and Satisfaction Guarantee QIAGEN guarantees the performance of all products in the manner described in our product literature The purchaser must determine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse QIAGEN will replace it free of charge or refund the purchase price We reserve the right to change alter or modify any product to enhance its performance and design If a QIAGEN product does not meet your expectations simply call your local Technical Service Department or distributor We will credit your account or exchange the product as you wish Separate conditions apply to QIAGEN scientific instruments service products and to products shipped on dry ice Please inquire for more information A copy of QIAGEN terms and conditions can be obtained on re
19. fication of a region containing codon 61 using the KRAS Pyro Kit Important points before starting M The HotStarTag DNA Polymerase in the PyroMark Master Mix requires an activation step of 15 min at 95 C E Set up all reaction mixtures in an area separate from that used for DNA purification adding template DNA to the PCR PCR product analysis or preparation of samples prior to Pyrosequencing analysis M Use disposable tips containing hydrophobic filters to minimize cross contamination Things to do before starting E Before opening the tubes with PCR primers centrifuge briefly to collect contents at the bottom of the tubes E Adjust the concentration of the control and sample DNA if necessary to 0 4 2 ng ul Procedure 1 Thaw all necessary reagents Mix well before use 2 Prepare a reaction mix for each PCR primer set according to Table 2 The reaction mix typically contains all of the components needed for PCR except the sample Prepare a volume of reaction mix greater than that required for the total number of PCR assays to be performed KRAS Pyro Handbook 09 2010 15 Table 2 Preparation of reaction mix for each PCR primer mix Component Volume reaction PyroMark PCR Master Mix 2x 12 5 ul CoralLoad Concentrate 10x 2 5 ul PCR Primer KRAS 12 13 or 1 ul PCR Primer KRAS 61 Water supplied 4 ul Total volume 20 pl 3 Mix the reaction mix thoroughly and dispense 20 pl into each PCR tube
20. for the KRAS Codon 61 assay as described in Appendix A Updated frequencies of mutations in the human KRAS gene in codon 12 13 and codon 61 are provided online by the Sanger Institute at www sanger ac uk genetics CGP cosmic KRAS Pyro Handbook 09 2010 For reliable results we recommend single peak heights above 30 RLU Set 30 RLU as the required peak height for passed quality in assay setup see Appendix A and the PyroMark Q24 User Manual The AQ Analysis results report should be used for documentation of allele quantification The numbers shown in the Pyrogram are rounded and do not show the exact quantification Reanalysis of samples with no mutation detected in nucleotide 35 Codon12 or 183 Codon 61 or with Check or Failed quality assessment We strongly recommend reanalyzing all samples with no mutation detected with the standard Sequence to Analyze as well as samples that received a Check or Failed quality assessment Check and Failed quality assessments may indicate a mutation in a position other than nucleotide 35 or 183 resulting in peak height deviations at reference dispensations For example a peak in any of the first 3 dispensations shows that a mutation is present at nucleotide 34 To reanalyze and target mutations at nucleotide 34 go to Analysis Setup and change Sequence to Analyze from GNTGRCGTAGGC to NGTGRCGTAGGC Click Apply and then click
21. instrument lid 7 Insert the USB stick containing the run file into the USB port at the front of the instrument Do not remove the USB port before the run is finished 8 Select Run in the main menu using the and screen buttons and press OK 9 Select the run file using the and screen buttons To view the contents of a folder select the folder and press Select To go back to the previous view press Back 10 When the run file is selected press Select to start the run 11 When the run is finished and the instrument confirms that the run file has been saved to the USB stick press Close 12 Remove the USB stick 13 Open the instrument lid 14 Open the cartridge gate and remove the reagent cartridge by lifting KRAS Pyro Handbook 09 2010 it up and pulling it out 23 15 Close the gate 16 Open the plate holding frame and remove the plate from the heating block 17 Close the plate holding frame and the instrument lid 18 Discard the plate and clean the cartridge as per the instructions in the product sheet supplied with the cartridge 19 Analyze the run according to Protocol 6 Analysis of a PyroMark Q24 Run page 25 24 KRAS Pyro Handbook 09 2010 Protocol 6 Analysis of a PyroMark Q24 Run This protocol describes the mutation analysis of a finished KRAS run using PyroMark Q24 Software Procedure 1 Insert the USB stick containing the processed run file into the
22. l 7 Proceed immediately with Protocol 4 Preparation of Samples Prior to Pyrosequencing Analysis on the PyroMark Q24 page 20 Sepharose beads sediment quickly Capturing of beads must take place immediately following agitation KRAS Pyro Handbook 09 2010 19 Protocol 4 Preparation of Samples Prior to Pyrosequencing Analysis on the PyroMark Q24 This protocol is for preparation of single stranded DNA and annealing of the sequencing primer to the template prior to Pyrosequencing analysis on the PyroMark Q24 Important points before starting B Before opening the tubes with sequencing primers centrifuge briefly to collect contents at the bottom of the tubes M Add the 2 different sequencing primers in the same pattern as predefined for the plate in the run setup see Protocol 1 Run Setup for the PyroMark Q24 System page 13 depending on the region of analysis codons 12 and 13 or codon 61 Things to do before starting M Place the PyroMark Q24 Plate Holder on a heating block at 80 C for use in step 17 M PyroMark Wash Buffer is supplied as a 10x concentrate Before using for the first time dilute to a 1x working solution by adding 225 ml high purity water to 25 ml 10x PyroMark Wash Buffer final volume of 250 ml Procedure 1 Dilute a sufficient amount of each sequencing primer Seq Primer KRAS 12 13 and Seq Primer KRAS 61 in PyroMark Annealing Buffer as shown in Table 6 Prepare a volume of diluted sequen
23. lyze if mutations are present in nucleotide 34 first position change the Sequence to Analyze to the following sequence NGTGRCGTAGGC Note Ensure the threshold for single peak height is set to 30 RLU A4 Manually enter the following Dispensation Order TACGACTCAGATCGTAG Figure 9 Histogram for codons 12 nucleotide 35 and 13 nucleotide 38 with the Sequence to Analyze GNTGRCGTAGGC 2 D ee Te o o Y o 9 oe ss petes c E ciem I 1 1 1 E 1 1 E 1 1 H 1 1 m H H B LJ G A T C G T A G T A c G A c T cC A Figure 10 Histogram for codons 12 nucleotide 34 and 13 nucleotide 38 with the Sequence to Analyze NGTGRCGTAGGC KRAS Pyro Handbook 09 2010 33 A5 Click the Analysis Parameters tab and increase Peak Height Threshold Required peak height for Passed quality to 30 A6 Click i in the toolbar and save the assay as KRAScodon 12 13 KRAS codon 61 Al Click in the toolbar and select New AQ Assay A2 Type the following sequence in Sequence to Analyze CTCDTGACCTG The most frequent mutations in codon 61 will be detected in nucleotide 183 third position with this sequence to analyze To analyze if mutations are present in nucleotide 182 second position change the Sequence to Analyze to the following sequence CTCTHGACCTG To analyze if mutations are present in nucleotide 181 first position change the Sequence to Analyze to the following sequence C
24. n base 2 of codon 12 nucleotide 35 indicated with an arrow KRAS Pyro Handbook 09 2010 29 Figure 7 Pyrogram trace obtained after analysis of samples with a GGT gt TGT mutation in base 1 of codon 12 nucleotide 34 indicated with an arrow with the Sequence to Analyze GNTGRCGTAGGC targeting base 2 in codon 12 nucleotide 35 A yellow color indicates that this sequence is unexpected and needs to be checked Figure 8 Pyrogram trace and result obtained after reanalysis of the sample in Figure 7 The mutation GGT gt TGT was reanalyzed with the Sequence to Analyze NGTGRCGTAGGC targeting base 1 in codon 12 nucleotide 34 EEE 30 KRAS Pyro Handbook 09 2010 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see also the Frequently Asked Questions page at our Technical Support Center www giagen com FAQ FAQbList aspx The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this handbook or sample and assay technologies for contact information see back cover or visit www qiagen com Refer to the PyroMark Q24 User Manual for general troubleshooting of the instrument Comments and suggestions Signals in the no template control negative control a Cross talk between Signal from one well is detected in a neighboring wells well If re running s
25. nstrument method according to the reagents and cartridge that will be used for the run see the instructions supplied with the products Plate ID Optional Enter ID of the PyroMark Q24 Plate Bar code Optional Enter a bar code number for the plate or if you have a bar code reader connected to your computer place the mouse cursor in the Barcode text box by clicking the box and scan the bar code Kit and Reagent ID Optional Enter the lot number for the KRAS Pyro Kit to be used The lot number can be found on the product label We recommend entering both the reagent ID and the kit ID so that any unexpected problems with the reagents can be traced Run note Optional Enter a note about the contents or purpose of the run Add assay files To add an assay to a well you can either M Right click the well and select Load Assay from the context menu B I Select the assay in the shortcut browser and click and drag the assay to the well A well is color coded according to the assay loaded to the well Enter sample IDs and notes To enter a sample ID or note select the cell and enter the text To edit a sample ID or note either select the cell the current contents will be selected or double click the cell 14 KRAS Pyro Handbook 09 2010 Protocol 2 PCR Using the PCR Reagents Supplied with the KRAS Pyro Kit This protocol is for PCR amplification of a region containing codon 12 and codon 13 and a separate PCR ampli
26. olve the enzyme mixture and the substrate mixture and also for the PCR and immobilization to Sepharose beads Protocols 2 and 3 Additional high purity water is required to dilute PyroMark Wash Buffer 10x M Ethanol 70 10 KRAS Pyro Handbook 09 2010 Important Notes General precautions The user should always pay attention to the following M Strict compliance with the user manual is required for optimal results Dilution of the reagents other than as described in this handbook is not recommended and will result in a loss of performance B I Use sterile pipet tips with filters for PCR setup M Store and extract positive materials specimens positive controls and amplicons separately from all other reagents and add them to the reaction mix in a spatially separated facility E Thaw all components thoroughly at room temperature 15 25 C before starting an assay E When thawed mix the components by pipetting repeatedly up and down or by pulse vortexing and centrifuge briefly Sample material All samples must be treated as potentially infectious material Specimen material is human DNA extracted from blood or formalin fixed paraffin embedded samples Samples from humans undergoing heparin treatment must not be used Blood samples that have been collected in tubes containing heparin as an anticoagulant should not be used Heparin affects the PCR DNA isolation The QIAGEN kits shown in Table 1 page 12 are r
27. on a result greater than 1 can be considered positive This level may vary considerably between replicates Table 7 LOD determined for specific mutations LOD COSMIC ID Mutation units V42 Codon 12 GGT GAT 2 2 521 GTT 1 0 7 520 TGT 2 1 516 AGT 1 9 517 GCT 2 3 522 CGT 1 8 518 Codon 13 GGC GAC 1 9 532 Codon 61 CAA as assayed in reverse orientation TTG GTG 2 8 554 TAG 3 1 553 TCG 3 5 552 ATG 2 6 555 TTC S 550 From the Catalogue of Somatic Mutations in Cancer available online at the Sanger Institute at www sanger ac uk genetics CGP cosmic t Lowest mutation level in a sample resulting in a measured frequency 2LOD For further explanation refer to the text above The KRAS Plug in Report algorithm was used to generate the LOD data Manual analysis as described in Protocol 6 page 25 may result in slightly different values 28 KRAS Pyro Handbook 09 2010 Representative results using the AQ analysis integral to the PyroMark Q24 System Representative Pyrogram results are shown in Figures 4 8 A 1 G 99 Figure 4 Pyrogram trace obtained after analysis of a sample with a normal genotype in codons 12 and 13 Figure 5 Pyrogram trace obtained after analysis of a sample with a normal genotype in codon 61 A 1 G 99 25 l l l l l l 1 1 l l l L l l l 1 l l l Figure 6 Pyrogram trace obtained after analysis of samples with a GGT gt GAT mutation i
28. ood Mini Kit Contents For 50 DNA preps 50 QlAamp MinElute Columns Proteinase K Buffers Collection Tubes 2 ml For 48 preps Reagent Cartridges Tissue Disposable Filter Tips Disposable Tip Holders Sample Tubes 2 ml Elution Tubes 1 5 ml Buffer G2 Proteinase K For collection fixation and stabilization of 10 samples 10 Prefilled Reagent Containers containing PAXgene Tissue Fix and PAXgene Tissue Stabilizer For 50 DNA preps PAXgene DNA Mini Spin Columns Processing Tubes Microcentrifuge Tubes Carrier RNA and Buffers to be used in conjunction with PAXgene Tissue Containers For 50 preps QlAamp Mini Spin Columns Buffers Reagents Tubes VacConnectors Cat no 56404 953034 765112 767134 61104 For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www giagen com or can be requested from QIAGEN Technical Services or your local distributor 38 KRAS Pyro Handbook 09 2010 Trademarks QIAGEN QlAamp QlAxcel BioRobot CoralLoad EZ1 HotStarTaq MinElute Pyro Pyrogram PyroMark Pyrosequencing QIAGEN Group Milli Q Millipore Corporation PAXgene PreAnalytiX GmbH Sepharose GE Healthcare Windows Microsoft Corporation Limited License Agreement Use of this product signifies the agreement of any purchaser or user of the KRAS P
29. quest and is also provided on the back of our invoices If you have questions about product specifications or performance please call QIAGEN Technical Services or your local distributor see back cover or visit www qiagen com 6 KRAS Pyro Handbook 09 2010 Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate material safety data sheets MSDSs These are available online in convenient and compact PDF format at www giagen com support MSDS aspx where you can find view and print the MSDS for each QIAGEN kit and kit component 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 6131 19240 Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of KRAS Pyro Kit is tested against predetermined specifications to ensure consistent product quality KRAS Pyro Handbook 09 2010 7 Introduction The KRAS Pyro Kit is used for quantitative measurements of mutations in codons 12 13 and 61 of the human KRAS gene The product consists of 2 assays one for detecting mutations in codons 12 and 13 and the second for detecting mutations in codon 61 Figure 1 The two regions are amplified separately by PCR and sequenced through the defined region Sequences s
30. s should be stored at 2 8 C upon arrival To minimize loss of activity it is advisable to keep both the enzyme mixture and the substrate mixture in the vials supplied Reconstituted enzyme and substrate mixtures are stable for at least 5 days at 2 8 C Reconstituted enzyme and substrate mixtures can be frozen and stored in their vials at 215 to 25 C Frozen reagents should not be subjected to more than 3 freeze thaw cycles Important Nucleotides should not be frozen The KRAS Pyro Kit is stable until the kit expiration date when stored under these conditions KRAS Pyro Handbook 09 2010 Product Use Limitations The KRAS Pyro Kit is intended for molecular biology applications This product is not intended for the diagnosis prevention or treatment of a disease All due care and attention should be exercised in the handling of the products We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines Technical Assistance At QIAGEN we pride ourselves on the quality and availability of our technical support Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of QIAGEN products If you have any questions or experience any difficulties regarding the KRAS Pyro Kit or QIAGEN products in general please do not h
31. the PyroMark Q24 Plate with the samples at 80 C for 2 min using the prewarmed PyroMark Q24 Plate Holder 18 Remove the PyroMark Q24 Plate from the plate holder and allow the samples cool to room temperature 15 25 C for 5 10 min 19 Proceed with Protocol 5 Running the PyroMark Q24 System page 23 22 KRAS Pyro Handbook 09 2010 Protocol 5 Running the PyroMark Q24 System This protocol describes the loading of PyroMark Gold Reagents into the PyroMark Q24 Cartridge and starting and finishing a run on the PyroMark Q24 For a detailed description about how to set up a run see the PyroMark Q24 User Manual Important point before starting M The Pre Run information report found in the Tools menu at run setup see Protocol 1 Run Setup for the PyroMark Q24 System page 13 provides information about the volume of nucleotides enzyme and substrate buffer needed for a specific assay Procedure 1 Dissolve each of the freeze dried enzyme and substrate mixtures in 620 pl water supplied 2 Load the PyroMark Q24 Cartridge with the appropriate volumes of nucleotides enzyme and substrate mixes 3 Open the cartridge gate and insert the filled reagent cartridge with the label facing out Push the cartridge in fully and then push it down 4 Ensure the line is visible in front of the cartridge and close the gate 5 Open the plate holding frame and place the plate on the heating block 6 Close the plate holding frame and the
32. ts containing e g Pyrograms and analysis quality Summary A puo a jq AA as ji Mutation codon 12 42 GGT AGT mmt B s Wild Wildtye 2 8 _ B4 jo Mutation codon 12 46 GGT AGT ais CEN CHR 777 RUNS m c RR COP EE MN NE cee MEE cs js Mutation codon 12 297 GGT GAT Jonon ca 39 Mutation codon 13 425 GGC GAC GDD See detailed results for further explanation Using the AQ analysis To analyze the run and get an overview of the results click one of the Analyze buttons Qui Analyze all wells Qus Analyze the selected well The analysis results allele frequencies and quality assessment are displayed above the variable position in the Pyrogram trace For more details on how to analyze a run see the PyroMark Q24 User Manual To generate a report select AQ Analysis Results or AQ Full Report in the menu Note The most frequent mutations in KRAS are found at nucleotide 35 second base of codon 12 Therefore the standard Sequence to Analyze for the KRAS Codon 12 and 13 assay as defined in the Analysis Setup addresses mutations at this position see Appendix A page 33 If a sample contains a mutation at nucleotide 34 first base of codon 12 the Sequence to Analyze can be changed to analyze also the mutation status at this position as described in Appendix A Similarly the Sequence to Analyze can be changed
33. urrounding the defined positions serve as normalization and reference peaks for quantification and quality assessment of the analysis FP Seq mm gt GTA GG Codons 12 amp 13 FPB wp Codon 61 TCCA GTT CTC lt Seq Figure 1 Illustration of the KRAS assay The sequence indicated is the analyzed sequence for a normal sample FP and FPB Forward PCR primers B indicates biotinylation RP and RPB Reverse PCR primers B indicates biotinylation Seq Sequencing primers Codons 12 and 13 are sequenced in the forward direction codon 61 in the reverse direction The product consists of a PCR primer mix and a sequencing primer for each assay The primers are delivered in solution Each vial contains 24 ul of each primer or primer mix Principle and procedure The workflow illustrates the assay procedure After PCR using primers targeting codons 12 13 and codon 61 the amplicons are immobilized on Streptavidin Sepharose High Performance beads Single stranded DNA is prepared and the corresponding sequencing primers anneal to the DNA The samples are then analyzed on the PyroMark Q24 System using a run setup file and a run file The KRAS Plug in Report should be used to analyze the run However the run can also be analyzed using the analysis tool integral to the PyroMark Q24 System The Sequence to Analyze can be then adjusted for detection of rare mutations after the run see Protocol 6 Analysis of a PyroMark Q24 Run
34. x A The KRAS Plug in Report can be obtained by e mail from pyro plugin giagen com This must only be done once before running the KRAS Pyro assay for the first time see Appendix A page 33 Procedure 1 Click U in the toolbar A new run file is created 2 Enter the run parameters see Run parameters page 14 3 Setup the plate by adding assays for both codons 12 13 and codon 61 to wells corresponding to the samples to analyze A negative control sample without DNA and the unmethylated control DNA provided are recommended as controls 4 When the run is set up and ready to run on the PyroMark Q24 System print a list of required volumes of enzyme mix substrate mix and nucleotides and the plate setup Select Pre Run Information from the Tools menu and when the report appears click 3 5 Close the run file and copy it to a USB stick supplied with the system using Windows Explorer The printed Pre Run Information can be used as a template for the sample setup see Protocol 3 Immobilization of PCR Products to Streptavidin Sepharose High Performance Beads page 18 To run the plate on PyroMark Q24 System see Protocol 5 Running the PyroMark Q24 System page 23 p gt LE LLLA LI iii SSS SSE Eee KRAS Pyro Handbook 09 2010 13 Run parameters Run name The name of the run is given when the file is saved Renaming the file also changes the name of the run Instrument method Select the i
35. yro Kit to the following terms 1 The KRAS Pyro Kit may be used solely in accordance with the KRAS Pyro Handbook and for use with components contained in the kit only QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included within this kit except as described in the KRAS Pyro Handbook and additional protocols available at www giagen com Other than expressly stated licenses QIAGEN makes no warranty that this kit and or its use s do not infringe the rights of third parties This kit and its components are licensed for one time use and may not be reused refurbished or resold QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated tnn By 9 The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court and shall recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and or its components For updated license terms see www giagen com 2010 QIAGEN all rights reserved www qiagen com Australia Orders 1 800 243 800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 1
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