Sample & Assay Technologies UGT1A1 Pyro® Handbook
Contents
1. Insert the USB stick containing the run file into the USB port at the front of the instrument Do not remove the USB stick before the run is finished Select Run in the main menu using the 4 and screen buttons and press OK 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 When the run file is selected press Select to start the run When the run is finished and the instrument confirms that the run file has been saved to the USB stick press Close Remove the USB stick Open the instrument lid it up and pulling it out Close the gate heating block Close the plate holding frame and the instrument lid UGTIAI Pyro Handbook 01 2011 21 Discard the plate and clean the cartridge as per the instructions in the product sheet supplied with the cartridge 22 Analyze the run according to Protocol 6 Analysis of a PyroMark Q24 Run page 26 UGTIAI Pyro Handbook 01 2011 25 Protocol 6 Analysis of a PyroMark Q24 Run This protocol describes the genotyping analysis of a finished UGT1A1 run using PyroMark Q24 Software Procedure 1 Insert the USB stick containing the processed run file into the 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 Softwa2. 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 sample DNA if necessary to 0 4 2 ng ul Procedure 1 Thaw all necessary components Mix well before use 2 Prepare a reaction mix for each PCR primer set according to Table 3 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 14 UGTIA1 Pyro Handbook 01 2011 Table 3 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 mix UGTIAI allele variant 28 or 1 0 ul PCR Primer mix UGT1A1 allele variant 6 Water supplied 4 0 ul Total volume 20 0 pl 3 Mix the reaction mix thoroughly and dispense 20 pl into each PCR tube 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 4 and mix thoroughly A negative control without template DNA should always be included Table 4 Preparation of PCR Component Volume reaction Reaction mix 20 ul Sam
3. o 5 Figure 8 Pyrogram trace obtained after analysis of samples with a G G genotype when analyzed for allele variant 6 28 UGTIAI Pyro Handbook 01 2011 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 FAQList 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 giagen 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 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 Low quality of genomic Low quality genomic DNA can cause the PCR to DNA fail Analyze PCR samples using an electrophoretic technique for example the QlAxcel System or agarose gel electrophoresis Check or failed result a Rare allele variants not Less
4. Sequence to Analyze 3 Manually enter the following Dispensation Order CATATATATGC 4 Click the Analysis Parameters tab and increase Peak Height Threshold Required peak height for Passed quality to 30 5 Click lel in the toolbar and save the assay as UGT1AT 28 Figure 9 Histogram for genotyping of UGT1A1 allele variant 28 UGTIAI 6 1 Click in the toolbar and select New AQ Assay 2 Type the sequence CRGAGCAT in Sequence to Analyze 3 Manually add the following Dispensation Order TCAGAGCA 4 Click the Analysis Parameters tab and increase Peak Height Threshold Required peak height for Passed quality to 30 5 Click in the toolbar and save the assay as UGTIAT 6 2 E EEHEHE 0 T C A G A G C A c 5 Figure 10 Histogram genotyping of UGTIAT allele variant 6 UGTIAI Pyro Handbook 01 2011 31 Appendix B Emptying the Waste Container and Troughs WARNING Hazardous chemicals AN 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 Dat
5. see Appendix B page 32 17 Heat 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 let 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 UGTIA1 Pyro Handbook 01 2011 Protocol 5 Running the PyroMark Q24 System This protocol describes the loading of reagents into the PyroMark Q24 Cartridge and starting and finishing a run on the PyroMark Q24 For detailed description on how to set up a run see the PyroMark Q24 User Manual Important point before starting E The Pre Run information report found in the Tools menu at run setup see Protocol 1 Run Setup for the PyroMark Q24 System page 12 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 each of water supplied 2 Mix by swirling the vial gently Do not vortex To ensure that the mixture is fully dissolved leave it at room temperature 15 25 C for 5 10 min Make sure that the solution is not turbid before filling the PyroMark Q24 Cartridge If the reagents are not to be used immediately place the reagent vials on ice or in a refrigerator 3 Allow the reagents and the PyroMark Q24 Cartridge to reach ambient temperatu
6. dATPa S dCTP dGTP dTTP and H O Accessories PyroMark Q24 Plate 24 well sequencing reaction 979201 100 plate PyroMark Q24 Cartridges for dispensing 979202 Cartridge 3 nucleotides and reagents PyroMark Vacuum Reusable filter probes for 979010 Prep Filter Probe PyroMark Vacuum Workstation 100 Q96 and Q24 PyroMark Control For installation check of system 979203 Oligo PyroMark Q24 For performance confirmation of 979204 Validation Oligo system Related products PyroMark Q24 Sequence based detection 9001514 platform for Pyrosequencing of 24 samples in parallel PyroMark Q24 Vacuum Workstation for 9001518 Vacuum Workstation preparing 24 samples in parallel 220 V from PCR product to 9001516 single stranded template 110 V 9001519 100 V UGTIAI Pyro Handbook 01 2011 33 Product Contents Cat no PyroMark Q24 Analysis software 9019062 Software QlAamp DNA FFPE For 50 DNA preps 50 QlAamp 56404 Tissue Kit 50 MinElute Columns Proteinase K Buffers Collection Tubes 2 ml EZ DNA Tissue Kit For 48 preps Reagent Cartridges 953034 48 Tissue Disposable Filter Tips Disposable Tip Holders Sample Tubes 2 ml Elution Tubes 1 5 ml Buffer G2 Proteinase K QlAamp DSP DNA For 50 preps QlAamp Mini Spin 61104 Blood Mini Kit Columns Buffers Reagents Tubes VacConnectors For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN
7. rere ELLEN LLL TA UGTIAI Pyro Handbook 01 2011 19 Table 7 Example of dilution of the sequencing primers Volume for Component Volume sample 9 1 reactions Seq Primer UGT1A1 28 or 0 8 ul 8 ul Seq Primer UGTIAT 6 PyroMark Annealing Buffer 24 2 ul 242 ul Total volume 25 0 pl 250 pl 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 System page 12 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 see Figure 2 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 vacuum tool by opening the vacuum switch 20 UGTIAI Pyro Handbook 01 2011 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 vacuum 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 the beads 6 Transfer
8. which contains other substances such as methanol or methylethylketone r E UGT1A1 Pyro Handbook 01 2011 9 Important Notes General precautions The user should always pay attention to the following M The components of this product are sufficient to perform the 24 reactions in 5 independent runs E Use sterile pipet tips with filters for PCR 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 M When thawed mix the components by pipetting repeatedly up and down or by pulse vortexing and centrifuge briefly W Failed results are not a basis for judgment of genotype 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 kits from QIAGEN shown in Table 1 are recommended for DNA purification from the indicated human sample types for use with the UGT1A1 Pyro Kit Carry out the DNA purification according to the instructions in the kit handbooks 10 UGTIAI Pyro
9. 6 36 37 39 45 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 the UGTIAT Pyro Kit is tested against predetermined specifications to ensure consistent product quality R36 38 Irritating to eyes and skin S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice S36 Wear suitable protective clothing S37 Wear suitable gloves S39 Wear eye face protection S45 In case of accident or if you feel unwell seek medical advice immediately show the label where possible 6 UGTIAI Pyro Handbook 01 2011 Introduction The UGTIAI Pyro Kit is used for genotyping of the allele variant 28 for distinguishing between 6 and 7 TA repeats and allele variant 6 for distinguishing between G and A genotype of the human UGTIAT gene The kit consists of two assays one for genotyping of allele variant 28 and the second for genotyping of allele variant 6 Figure 1 The two regions are amplified separately by PCR and sequenced through the defined region Sequences surrounding the defined positions serve as normalization and reference peaks for genotyping and quality assessment of the analysis The product consists of a PCR primer mix and a sequencing primer for each as
10. Handbook 01 2011 Table 1 DNA purification kits recommended for use with the UGTIAT Pyro Kit Catalog number Sample material Nucleic acid isolation kit QIAGEN Blood QlAamp DSP DNA Blood Mini Kit 61104 Paraffin embedded QlAamp DNA FFPE Tissue Kit 50 56404 tissue EZ1 DNA Tissue Kit 48 953034 Recommended plate mixers The plate mixers shown in Table 2 are recommended for use with the UGT1A1 Pyro Kit Table 2 Plate mixers recommended for use with the UGT1A1 Pyro Kit Manufacturer Product Catalog number Basic mixer 5355 000 011 Eppendorf Thermoblock for MTP 5363 000 012 Adapter plate for 96 x 0 2ml PCR tubes to insert in blocks for 5363 007 009 microtiter plates ven Bsp 51410 ariomag Teleshake H P 4 115 V 51410 U Labortechnik 51110 einer Variomag Monoshake 115V 51110 U Controls Human control DNA is included in the kit as a positive control for PCR and sequencing reactions In addition a negative control without template DNA should always be included UGT1A1 Pyro Handbook 01 2011 11 Protocol 1 Run Setup for the PyroMark Q24 System Things to do before starting E Create an Assay Setup as described in Appendix A This needs to be done only once before running the UGT1A1 assay for the first time see Appendix A page 31 Procedure 1 Click in the toolbar A new run file is created 2 Enter the run parameters see Run parameters page 13 3 Set up the plate by ad
11. January 2011 UGTIAI Pyro Handbook For genotyping of the allele variants 28 and 6 of the human UGTIAT 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 5 Safety Information 5 Quality Control 6 Introduction 7 Principle and procedure 7 Equipment and Reagents to Be Supplied by User 9 Important Notes 10 General precautions 10 Sample material 10 DNA isolation 10 Recommended plate mixers 11 Controls 11 Protocols B 1 Run Setup for the PyroMark Q24 System 12 B 2 PCR Using the Reagents Supplied with the UGT1A1 Pyro Kit 14 B 3 Immobilization of PCR Products to Streptavidin Sepharose High Performance Beads 17 E 4 Preparation of Samples Prior to Pyrosequencing Analysis on the PyroMark Q24 System 19 E 5 Running the PyroMark Q24 System 23 B 6 Analysis of a PyroMark Q24 Run 26 Troubles
12. NA 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 12 4 Add 10 pl of 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 12 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 UGTIAI Pyro Handbook 01 2011 17 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 Manual 7 Proceed immediately with Protocol 4 Preparation of Samples Prior to Pyrosequencing Analysis on the PyroMark Q24 System page 19 Sepharose beads sediment quickly Capturing of the beads must take place immediately following agitation If more than 1 min has elapsed since the plate or strips was agitated agitate again for 1 min before capturing the beads Lon K M E 18 UGTIAI Pyro Ha
13. a 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 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 Procedure 1 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 2 Discard any solutions left in the troughs 3 Rinse the troughs with high purity water or replace them if necessary 4 Empty the waste container 5 The cap can be removed without disconnecting the tubing 6 If the vacuum workstation must be cleaned for example due to dust or spillage follow the instructions in the PyroMark Q24 User Manual 32 UGTIA1 Pyro Handbook 01 2011 Ordering Information Product Contents Cat no UGTIAI Pyro Kit For 24 reactions on PyroMark 970540 24 Q24 Systems Seq Primers PCR Primers Human Control DNA PyroMark PCR Master Mix CoralLoad Concentrate PyroMark Binding Buffer PyroMark Annealing Buffer PyroMark Denaturation Solution PyroMark Wash Buffer Enzyme Mixture Substrate Mixture
14. acilitate 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 2011 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 10 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 Jap
15. an 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 7050 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 E Sample amp Assay Technologies
16. ding assays for allele variant 28 and allele variant 6 to wells corresponding to the samples to analyze A negative sample without DNA and a control DNA are recommended as controls 4 When the run is set up and ready to run on the PyroMark Q24 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 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 set up see Protocol 3 Immobilization of PCR Products to Streptavidin Sepharose High Performance Beads page 17 To run the plate on the PyroMark Q24 see Protocol 5 Running the PyroMark Q24 System page 23 A 12 UGT1A1 Pyro Handbook 01 2011 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 instrument method according to the 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 En
17. frequent number of TA repeats not covered defined in the assay by the Assay setup or by the standard Sequence setup to Analyze can result in changed reference pattern and Check or Failed quality assessment 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 c High peak height The Pyrogram should be carefully compared to deviation warning at the histogram which can be displayed by a right dispensation x click in the Pyrogram window UGTIAI Pyro Handbook 01 2011 29 Comments and suggestions High background Incorrect storage of Store nucleotides at 2 8 C Storage at nucleotides 20 C can cause an increase in the background No signals in positive controls a 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 UGTIAT Pyro Kit reagents according stored or diluted to the instructions supplied 30 UGTIAI Pyro Handbook 01 2011 Appendix A Setting Up UGT1A1 Pyro Assays Set up the assay for UGT1A1 allele variants by using the PyroMark Q24 Software Before running the UGT1A1 assay for the first time the assay file needs to be set up as described below Procedure UGTIAI 28 1 Click in the toolbar and select New AQ Assay 2 Type the sequence ATATAT AT GGCA in
18. hooting Guide 29 Appendix A Setting Up UGT1A1 Pyro Assays 31 Appendix B Emptying the Waste Container and Troughs 32 Ordering Information 33 UGTIAI Pyro Handbook 01 2011 3 Kit Contents UGT1A1 Pyro Kit box 1 2 UGTIAI Pyro Kit 24 Catalog no 970540 Number of reactions 24 PCR Primer mix UGT1A1 28 24 ul PCR Primer mix UGT1A1 6 24 ul Seq Primer UGT1A1 28 24 ul Seq Primer UGTIAT 6 24 ul PyroMark PCR Master Mix 2x 850 ul CoralLoad Concentrate 10x 1 2 ml H O 3x1 9 ml Human Control DNA 10 ng ul 100 ul Pyro 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 vial Substrate Mixture 1 vial dATPaS 1180 ul dCTP 1180 ul dGTP 1180 ul dTTP 1180 ul Handbook 1 Contains sodium hydroxide UGTIAI Pyro Handbook 01 2011 Shipping and Storage The UGTIAI Pyro Kit is shipped in two boxes The UGT1A1 Pyro Kit box 1 2 is shipped on dry ice PyroMark PCR Master Mix CoralLoad Concentrate 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 components should be stored at 2 8 C upon arrival To minimize loss of activity it is advisable to kee
19. kit handbooks and user manuals are available at www giagen com or can be requested from QIAGEN Technical Services or your local distributor EA 34 UGT1A1 Pyro Handbook 01 2011 Trademarks QIAGEN QlAamp QlAxcel CoralLoad EZ1 HotStarTaq MinElute Pyro Pyrogram PyroMark Pyrosequencing QIAGEN Group Milli Q Millipore Corporation Sepharose GE Healthcare Windows Microsoft Corporation Limited License Agreement Use of this product signifies the agreement of any purchaser or user of the UGT1A1 Pyro Kit to the following terms 1 The UGTIAI Pyro Kit may be used solely in accordance with the UGTTAT Pyro Kit 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 UGTTAT Pyro Kit Handbook and additional protocols available at www qiagen 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 C exp i09 NS The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or f
20. ndbook 01 2011 Protocol 4 Preparation of Samples Prior to Pyrosequencing Analysis on the PyroMark Q24 System 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 System Important points before starting E Before opening the tubes with sequencing primers centrifuge briefly to collect contents at the bottom of the tubes E 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 12 depending on the region of analysis allele variant 28 or allele variant 6 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 add high purity water to 25 ml 10x PyroMark Wash Buffer to achieve a final volume of 250 ml and obtain a 1x working solution Note The 1x PyroMark Wash Buffer working solution is stable at 2 8 C until the marked expiration date Procedure 1 Dilute a sufficient amount of each sequencing primer Seq Primer UGT1A1 28 and Seq Primer UGTIAT 6 in PyroMark Annealing Buffer as shown in Table 7 Prepare a volume of diluted sequencing primer greater than that required for the total number of samples to be sequenced for the number of samples one extra
21. ol 4 Nc ae PyroMark Q24 run Protocol 5 Y Analysis of PyroMark Q24 run Protocol 6 Y Report 8 UGTIAI Pyro Handbook 01 2011 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 10 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 varies depending on region see Ordering Information page 33 Plate mixer for immobilization to beads see Recommended plate mixers page 11 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 provided in the product to dissolve the Enzyme Mixture and the Substrate Mixture for immobilization and for PCR additional high purity water is required to dilute PyroMark Wash Buffer 10x M Ethanol 70 Do not use denatured alcohol
22. p 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 UGTIAI Pyro Kit is stable until the kit expiration date when stored under these conditions Product Use Limitations The UGTIAI Pyro Kit is intended for molecular biology applications This product is not intended for the diagnosis prevention or treatment of a disease 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 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 qiagen com Support MSDS aspx where you can find view and print the MSDS for each QIAGEN kit and kit component ieee 3 UGTIAI Pyro Handbook 01 2011 5 The following risk and safety phrases apply to components of the UGT1A1 Pyro Kit PyroMark Denaturation Solution Contains sodium hydroxide Irritant Risk and safety phrases R36 38 2
23. ple DNA 5 ul Total volume 25 pl eee SSE UGTIAI Pyro Handbook 01 2011 15 5 Program the thermal cycler according to the manvfacturer s instructions using the conditions outlined in Table 5 Table 5 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 7220 Number of cycles 42 Final extension 5 min 72 C 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 17 16 UGTIAI Pyro Handbook 01 2011 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 PyroMark Q24 System Things to do before starting E 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 6 Prepare a volume 1096 greater than that required for the total number of reactions to be performed Table 6 Master mix for D
24. re 20 25 C 4 Place the PyroMark Q24 Cartridge with the label facing you 5 Load the PyroMark Q24 Cartridge with the appropriate volumes of nucleotides enzyme and substrate mixes according to Figure 4 Make sure that no air bubbles are transferred from the pipet to the cartridge 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 UGTIAI Pyro Handbook 01 2011 23 11 12 13 14 15 16 Open the cartridge gate and remove the reagent cartridge by lifting 17 18 Open the plate holding frame and remove the plate from the 19 20 24 4 s O micis Label He Figure 4 Illustration of the PyroMark Q24 Cartridge as seen from above The annotations correspond to the label on the reagent vials Add enzyme mixture E substrate mixture S and nucleotides A T C G according to the volume information given in the Pre Run information report found in the Tools menu at run setup 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 Ensure the line is visible in front of the cartridge and close the gate Open the plate holding frame and place the plate on the heating block Close the plate holding frame and the instrument lid
25. re either by selecting Open in the File menu or by double clicking the file 6 in the shortcut browser 4 To analyze the run and get an overview of the results click one of the Analyze buttons n Analyze all wells Qus Analyze the selected well For more details on how to analyze a run see the PyroMark Q24 User Manual 5 To generate a report select SNP Full Report or SNP Overview Report in the Reports menu 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 page 31 and the PyroMark Q24 User Manual The Pyrogram should always be compared to the histogram which can be displayed by a right click in the Pyrogram window The measured peaks should match the height of the histogram bars 26 UGTIAI Pyro Handbook 01 2011 Representative results Representative Pyrogram results are shown in Figures 5 8 Figure 5 Pyrogram trace obtained after analysis of a sample with TA6 TA6 genotype when analyzed for allele variant 28 200 150 100 50 Figure 6 Pyrogram trace obtained after analysis of a sample with TA TA6 TA7 genotype when analyzed for allele variant 28 250 200 150 100 o ui oo Figure 7 Pyrogram trace obtained after analysis of a sample with TA TA TA7 TA7 genotype when analyzed for allele variant 28 UGTIAI Pyro Handbook 01 2011 27 150 100 1 1 4 1
26. say The primers are delivered in solution Each vial contains 24 ul of each primer or primer mix Note The allele variant 28 is sequenced in reverse orientation and the allele variant 6 in forward orientation UGT1A1 28 CGG TA TATATA d dignus RP Seq Seq c gt CA GGAGCA UGT1A1 6 Figure 1 Illustration of the UGT1A1 assays The sequence indicated is the analyzed sequence with polymorphic nucleotides indicated by square brackets or slash Part of the TA repeats analyzed with the UGT1A1 28 assay are covered by the sequencing primer FP FPB Forward PCR primers B indicates biotinylation RP RPB Reverse PCR primers B indicates biotinylation Seq Sequencing primers Principle and procedure The workflow on page 8 illustrates the assay procedure After PCR using primers targeting allele variants 28 and 6 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 see Protocol 6 Analysis of a PyroMark Q24 Run page 26 and Appendix A page 31 UGTIAI Pyro Handbook 01 2011 7 Workflow of UGT1A1 Pyro procedure Assay and run setup Sample preparation Assay file setup Appendix A PCR Protocol 2 Y i Immobilization Protocol 3 Y Run file setup Protocol 1 Preparation of samples Protoc
27. ter the lot number for the UGTIAT Pyro Kit to be used The lot number can be found on the product label Note 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 E 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 UGTIAI Pyro Handbook 01 2011 13 Protocol 2 PCR Using the Reagents Supplied with the UGTIAT Pyro Kit This protocol is for PCR amplification of a region for genotyping of allele variant 28 and a separate PCR amplification of a region for genotyping of allele variant 6 using the UGT1A1 Pyro Primers Important points before starting M The HotStarTag DNA Polymerase in the PyroMark PCR Master Mix requires an activation step of 15 min at 95 C E Setup 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
28. the vacuum tool to the trough containing 40 ml 7096 ethanol Figure 2 Flush the filter probes for 5 s 7 Transfer the tool to the trough containing 40 ml Denaturation Solution Figure 2 Flush the filter probes for 5 s 8 Transfer the vacuum tool to the trough containing 50 ml Wash Buffer Figure 2 Flush the filter probes for 10 s 9 Raise the vacuum tool up and back beyond 90 vertical for 5 s to drain liquid from the filter probes Figure 3 Figure 3 Illustration of the vacuum tool raised to beyond 90 vertical 10 While the vacuum tool is held over the PyroMark Q24 Plate close the vaccum 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 vacuum tool to the trough containing high purity water Figure 2 and agitate the tool for 10 s 13 Wash the filter probes by lowering the probes into high purity water Figure 2 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 Figure 3 15 Close the vacuum switch on the tool Off and place the tool in the Parking P position UGTIAI Pyro Handbook 01 2011 21 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
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