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1 V2, 2014/07 1 OF 40

2 IMPORTANT NOTES AND UPDATES V2, 2014/07 2 OF 40

3 V2, 2014/07 3 OF 40

4 CONTENT 1 Key to symbols 6 2 Kit content 7 3 Shipping and Storage 7 4 Technical assistance 7 5 Warning and precautions 8 6 Principle 10 7 Procedure 11 8 Equipment and Reagents 12 9 Important notes before starting Protocols 16 Protocol 1: Amplification procedure 16 Protocol 2: Clean-up of PCR products 20 Protocol 3: Sequencing reaction 22 Protocol 4: Sequencing product clean-up 24 Protocol 5: Genetic Analyzer Appendix A: High-resolution HLA typing Appendix B: Contamination control Troubleshooting guide Limited license agreement 34 Ordering information 40 V2, 2014/07 4 OF 40

5 Disclaimer GenDx has made every effort to ensure that this Instructions For Use (IFU) is accurate. GenDx disclaims liability for any inaccuracies or omissions that may have occurred. Information in this IFU is subject to change without notice. GenDx assumes no responsibility for any inaccuracies that may be contained in this IFU. GenDx reserves the right to make improvements to this IFU and/or to the products described in this IFU, at any time without notice. If you find information in this manual that is incorrect, misleading, or incomplete, we would appreciate your comments and suggestions. Please send them to Copyright This publication, including all photographs, illustrations, is protected under international copyright laws, with all rights reserved. Neither this manual, nor any of the material contained herein, may be reproduced without written consent of the author. Copyright 2014 V2, 2014/07 5 OF 40

6 1 KEY TO SYMBOLS Material number Components Batch code/lot Number Catalogue Number Consult Instructions For Use Contains reagents for N tests Legal Manufacturer o -20 C. Store at -20 C Store at -20 C Store at -20 C Add liquid V2, 2014/07 6 OF 40

7 2 KIT CONTENT Each kit contains reagents sufficient for 96 reactions. For full description of kit content, please see the kit content label on the box or visit our website ( 3 SHIPPING AND STORAGE SBTessenz HLA kits are: Shipped at ambient temperature and should be stored at -20 C upon arrival. Stable until the kit expiration date, as depicted on box label, when stored at -20 C. 4 TECHNICAL ASSISTANCE For technical assistance and more information: support@gendx.com Website: Phone: or contact your local GenDx distributor ( V2, 2014/07 7 OF 40

8 5 WARNING AND PRECAUTIONS Product Use Limitations For Research Use Only. Not for use in diagnostic procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease. Every attempt has been made to validate all amplification and sequencing primers used in SBTessenz with DNA samples typed by molecular methods. Due to the limited access to such reference materials, some rare alleles may not have been tested with SBTessenz. To ensure the best performance, please use the SBTessenz kits with the materials, reagents, and equipments recommended in section 8 Equipment and Reagents to be supplied by User. Use of materials other than specified, must be validated by user! Reconstitution or dilution of primers in volumes other than described in this IFU can lead to performance errors and is strongly discouraged! Please take special note of Appendix B: Contamination control. V2, 2014/07 8 OF 40

9 Before implementing SBTessenz in your laboratory, please perform a validation for Sequencing-Based Typing methods using known molecular typed samples. Such samples may be obtained from the International Workshop Reference Cell Panel and the UCLA DNA Reference Panel. Safety Information Please read the Safety Information and Important Notes sections of QIAGEN s Taq PCR Core kit Handbook and of the BigDye Terminator protocol manual before beginning the procedure. 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. V2, 2014/07 9 OF 40

10 6 PRINCIPLE The SBTessenz assay targets the gene regions that code for the antigen recognition site. The assay set-up provides an efficient combination of generic and group-specific sequencing primers (GSSPs) to accomplish enhanced ambiguity resolution in one single round of sequencing. For class I (HLA-A, -B and -C) exon 2 and 3 are amplified and sequenced. For class II (HLA-DQB1 and -DRB1) exon 2 is amplified and sequenced. GSSP results The SBTesssenz assay approach includes GSSPs in the first round of sequencing. The result to be expected from the GSSP reaction depends on the characteristic of the alleles present in the sample. In some cases the GSSP resolves the ambiguity (homozygous sequence). In other cases, the GSSP matches both alleles present (heterozygous sequence), or doesn t match any of the alleles present (rejected sequence). The latter can also result in detection of sequences in unexpected genomic regions. Importantly, GSSP results cannot give wrong typing results and GSSP reactions can be excluded from analysis when the outcome does not contribute to resolvability. V2, 2014/07 10 OF 40

11 7 PROCEDURE SBTessenz consists of two types of 96-well primer plates: 1) The 96-well AMP primer plate: each well contains dried amplification primer mix sufficient for one HLA locus-specific amplification by thermal cycling using the Taq polymerase PCR system (Qiagen). 2) The 96-well SEQ primer or Group-Specific Sequencing primer (GSSP) plate: each well contains dried sequencing primer mix sufficient for one sequencing reaction using the BigDye Terminator sequencing chemistry v1.1 (Life Technologies). Prior to sequencing, the PCR products are treated with ExoI/SAP to degrade unincorporated primers and dephosphorylase residual nucleotides. The final sequencing reactions are purified using Sephadex G-50 columns to remove unincorporated sequencing primers and residual nucleotides. Denatured samples are loaded on an automated Genetic Analyzer. Use the Genetic Analyzer s analysis software to process collected raw data and use SBTengine or other suitable HLA typing software to analyze the sequence files. V2, 2014/07 11 OF 40

12 8 EQUIPMENT AND REAGENTS TO BE SUPPLIED BY USER The performance of the SBTessenz kits is validated with the reagents and equipment described in this protocol. Use of other reagents or equipment requires user validation. SBTessenz products have been tested on Applied Biosystems GeneAmp PCR System 9700 using the Taq PCR Core kit. Other thermal cyclers or long extension PCR kits may require different profiles, and may require user validation. The following list includes the essential equipment and reagents. General Thermal cycler Micro centrifuge Vortex Deionised water Centrifuge (rotor and adapters for 96 well microtiter plates) V2, 2014/07 12 OF 40

13 HLA locus amplification QIAGEN Taq PCR Core kit PCR tubes (use thin-walled 0.2 ml PCR tubes recommended by the manufacturer of your thermal cycler) Agarose gel electrophoresis system Sequencing of HLA loci Exonuclease I and Shrimp Alkaline Phosphatase 1.5 or 2 ml micro centrifuge tubes BigDye Terminator v1.1 Cycle sequencing kit or BigDye Terminator v3.1 cycle sequencing kit Clean-up and analysis of HLA sequencing products Sephadex G-50 Superfine Multiscreen 45 l Column Loader Multiscreen Column Loader Scraper clear Multiscreen-HV Multiscreen Centrifuge Align Frame Blue EU Frosted Sub Skirted Thin-wall 96x0.2 ml plates Capillary sequencer (e.g., ABI ABI PRISM 3100 or Applied Biosystems 3130 Genetic Analyzers, or ABI PRISM 3700 or Applied Biosystems 3730 DNA Analyzers, Applied biosystems) SBTengine software to analyze sequence files and to create HLA typing reports V2, 2014/07 13 OF 40

14 9 IMPORTANT NOTES BEFORE STARTING Sample Preparation Purified DNA should have an A260/A280 ratio between 1.7 and 1.9. If necessary, DNA should be diluted in Nuclease Free H2O before use. The optimal amount of template DNA to use in the reaction is 40 ng. However, template DNA in the range of ng (in 1 2 µl) can be used without affecting results. To streamline the process, validate your DNA purification procedure so you can use a set volume corresponding to ng DNA. Blood samples should be collected in ACD or EDTA anticoagulant tubes. Do NOT use heparinized samples, as this has an inhibitory effect on a PCR. Assay set-up Set up all reactions on ice. Prepare a volume of reaction mix at least 10% greater than required for the total number of assays to be performed. V2, 2014/07 14 OF 40

15 SBTessenz plate preparation 1. Spin all 96-well primer plates briefly at maximum RPM before use. 2. The AMP and SEQ primers are already present in the 96-well primer plates. 3. The primers are visible as orange pellets. V2, 2014/07 15 OF 40

16 10 PROTOCOLS PROTOCOL 1: AMPLIFICATION PROCEDURE 1. Set up all reactions on ice. 2. HLA locus-specific amplification is performed by thermal cycling using the SBTessenz HLA AMP plate and the Taq PCR CORE kit (Qiagen). Each well contains dried amplification primers sufficient for one PCR. 3. Prepare amplification (AMP) Plate Mix by mixing all PCR components, except for DNA. Centrifuge all PCR reagents before starting. 4. Pipet sufficient volumes for one AMP plate (96 samples +10% pipetting volume) according to Table 1 and 1A (DQB1). For HLA-DQB1 the addition of Q solution is required. 5. Mix the AMP Plate Mix thoroughly, and centrifuge briefly. 6. Dispense appropriate volumes into each well of the 96-well HLA AMP plate. V2, 2014/07 16 OF 40

17 Table 1: Composition of AMP Plate Mix for locus-specific amplification. HLA- A, B, C and DRB1 Component N=1 N=105^ Nuclease free H2O μl μl PCR buffer (10x)* 1 μl 105 μl dntp mix 0.25 μl μl Taq DNA polymerase (5U/μl) 0.06 μl 6.3 μl AMP primer (dried pellet) - - Template DNA 1-2 μl Variable Total Volume 10 μl Table 1A: Composition of AMP Plate Mix for locus-specific amplification. HLA- DQB1 Component N=1 N=105^ Nuclease free H2O μl μl PCR buffer (10x) * 1 μl 105 μl dntp mix 0.25 μl μl Taq DNA polymerase (5U/μl) 0.06 μl 6.3 μl Q-solution (5x) 2 μl 210 μl AMP primer (dried pellet) - - Template DNA 1-2 μl Variable Total Volume 10 μl ^96 samples + 10% pipetting volume * Use the 10x PCR buffer without CoralLoad, primer mix includes loading dye. V2, 2014/07 17 OF 40

18 7. Prepare fresh template DNA solutions in nucleasefree water. 8. Add template DNA to each well containing the AMP Plate Mix. The final volume must be 10 μl. The optimal amount of template DNA to use in the reaction is 40 ng. Minimize the time between mixing the PCR reagents with the DNA and the initiation of the thermal cycling. 9. Seal the 96-well HLA AMP plate using the provided adhesive PCR film, mix thoroughly and centrifuge briefly. 10. Program the thermal cycler according to the manufacturer s instructions, using the conditions outlined in Table 2. Important: For a simplified hot-start, place the tubes in a thermo cycler that is preheated to 95 C and start the cycling program. 11. PCR products must be confirmed by agarose gel electrophoresis. Prepare a 1.5% (w/v) agarose gel according to your laboratory protocol. Load the gel with 3 μl of PCR product. Gel can be loaded without addition of loading dye. V2, 2014/07 18 OF 40

19 Table 2. Cycling protocol for amplification. Step Temp Time Initial denaturation 95 C 3 min 3-step cycling Denaturation 95 C 30 sec Annealing 62 C 30 sec Elongation 72 C 1 min 35 cycles Final elongation 72 C 10 min Cooling 15 C Table 3: Approximate size of PCR products Locus A B C DQB1 DRB1 Expected size 1.0 kb 1.0 kb 1.2 kb 0.6 kb 0.5 to 0.8 kb V2, 2014/07 19 OF 40

20 PROTOCOL 2. CLEAN-UP OF PCR PRODUCTS 12. Set up all reactions on ice. 13. Prepare an ExoI/SAP Master Mix in a clean microfuge tube according to Table Add 1.5 μl ExoI/SAP Master Mix to each of the PCR products. 15. Gently shake the mixture to produce a homogeneous mixture and centrifuge briefly. 16. Program the thermal cycler according to the manufacturer s instructions, using the conditions outlined in Table 5. V2, 2014/07 20 OF 40

21 Table 4. Composition of Exol/SAP reaction mix for amplicon clean-up. Component N = 1 N = 105 Exo I (20U/ μl) 0.5 μl 53 μl SAP (1 U/ μl) 1 μl 106 μl Total volume 1.5 μl 159 μl Table 5. ExoI/SAP cycling protocol for amplicon clean-up. Step Temp Time Enzyme activation 37 C 30 min Enzyme deactivation 80 C 20 min Cooling 15 C V2, 2014/07 21 OF 40

22 PROTOCOL 3. SEQUENCING REACTION 18. Set up all reactions on ice. 19. Dilute the PCR products by adding 8µl deionised water. 20. Briefly mix and centrifuge all sequencing reagents before use. 21. The sequencing primers are already present in the 96-well SEQ/GSSP plate. Prepare a sequencing (SEQ) Plate Mix sufficient for the total number of sequencing assays to be performed, according to Table Gently mix the SEQ Plate Mix to produce a homogeneous reaction and then centrifuge briefly. 23. Dispense 9 μl of the SEQ Plate Mix into each well of the 96-well SEQ plate. 24. Add 1 μl purified (diluted) PCR product to each well containing the SEQ Plate Mix. 25. Place the samples in the thermal cycler and start cycling using the conditions outlined in Table 7. V2, 2014/07 22 OF 40

23 Table 6. Composition of SEQ Plate Mix for sequencing of HLA loci. Component N = 1 N =105 Nuclease free H2O 6.5 μl μl BDT Buffer (5x) 1.5 μl μl BDT Ready Reaction Premix (2.5x) 1 μl 105 μl SEQ primer (dried pellet) - - Total volume 9 μl 945 μl Table 7. Cycling protocol for sequencing. Step Temp Time Initial denaturation 96 C 10 sec 3-step cycling Denaturation 96 C 10 sec Annealing 50 C 10 sec Elongation 60 C 2 min 25 cycles Cooling 15 C V2, 2014/07 23 OF 40

24 PROTOCOL 4. SEQUENCING PRODUCT CLEAN-UP by Sephadex 26. Prepare Sephadex at least 3 hours before use. 27. Fill the wells of the Multiscreen (MS) 45 μl Column Loader with Sephadex G-50 and remove the remaining Sephadex G-50 by scraping with the MS Column Loader Scraper Clear. 28. Place a MS-HV plate upside-down on top of the filled MS 45 μl Column Loader and turn it upsidedown to fill the MS-HV plate with Sephadex G Add 300 μl deionised water to each well (avoid air bubbles) and let the Sephadex steep. The Sephadex should steep at least 3 hours before use but no longer than overnight. Store the Sephadex at 4 C when steeping overnight. Make sure the columns do not dry out! 30. Put the MS-HV plate containing the steeped Sephadex G-50 on a 96-wells microtiter plate placing the MS Centrifuge Align Frame Blue in between, and spin down at 750 x g for 5 min. V2, 2014/07 24 OF 40

25 31. Replace the 96-wells microtiter plate and the MS Centrifuge Align Frame Blue by the Genetic Analyzer run plate (e.g. EU Frosted Subskirted Thinwall 96 x 0.2 ml. plate). Discard the flow-through and store the 96-wells microtiter plate for re-use. 32. Pipet 10 μl deionised water, followed by 10 μl of sequence sample on the MS-HV plate, in the middle of the well. 33. Spin down at 750 x g for 5 min. to collect the flowthrough in the Genetic Analyzer run plate. Continue to the next protocol for immediate processing of the sequencing products on the Genetic Analyzer, or cover the Genetic Analyzer run plate containing the sequencing products with an adhesive seal and store at -20 C for long-term storage. V2, 2014/07 25 OF 40

26 PROTOCOL 5. GENETIC ANALYZER 34. Seal the Genetic Analyzer run plate with the grey flexible cover. 35. Load the Genetic Analyzer run plate containing the samples into the thermal cycler, heat the samples for 2 min. at 95 C and subsequently put them on ice until loading in the Genetic Analyzer. 36. Run the Genetic Analyzer according to the manufacturer s instructions. 37. Use the standard parameters to run your sequencer for your POP version and capillary length. 38. Process the collected raw sequence data with the SBTengine software. Other HLA typing software may require user validation. V2, 2014/07 26 OF 40

27 11 APPENDIX A. HIGH-RESOLUTION HLA TYPING As SBTessenz targets the gene regions that code for the antigen recognition site, the typing results of SBTessenz will be at an intermediate resolution level. For highresolution levels GenDx provides SBTexcellerator kits. These kits cover more exons and can solve more ambiguities by using additional sequencing primers and GSSPs. SBTexcellerator kits are available as RUO and CE-IVD for HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1, DPB1, and -G (HLA-G RUO only). For a complete overview please visit V2, 2014/07 27 OF 40

28 12 APPENDIX B. CONTAMINATION CONTROL IMPORTANT: It is extremely important to include at least one negative control in every PCR setup that lacks template nucleic acid to detect possible contamination. General Physical Precautions Separate the working areas for setting up the PCR amplification mix and DNA handling, including the addition of starting template, PCR product analysis, or plasmid preparation. Ideally, use separate rooms. Use a separate set of pipettes for the PCR amplification mix. Use of pipette tips with hydrophobic filters is strongly recommended. Prepare and freeze small aliquots of primer solutions and dntp mix. Use of fresh Nuclease Free H2O is strongly recommended. In case of contamination, laboratory benches, apparatus, and pipettes can be decontaminated by cleaning them with a 1% Trigene disinfectant. Afterwards, the benches and pipettes must be rinsed thoroughly with Nuclease Free H2O. V2, 2014/07 28 OF 40

29 General Chemical Precautions PCR stock solutions can also be decontaminated using UV light. This method is laborious however, and its efficiency is difficult to control and cannot be guaranteed. We recommend storing solutions in small aliquots and using fresh aliquots for each PCR. Another approach to prevent amplification of contaminating DNA is to treat individual reaction mixtures with DNAse or restriction enzymes that cut between the binding sites of the amplification primers used, before adding the template DNA sample. V2, 2014/07 29 OF 40

30 13 TROUBLESHOOTING GUIDE Absence of PCR product or weak PCR product Taq polymerase was not added to the amplification mix or not mixed properly when added Thermal cycler failure Repeat amplification paying attention to the addition and mixing of the polymerase with the amplification mix. Check the cycler run history. Perform maintenance or reprogram the thermal cycler. DNA concentration not optimal Re-quantify DNA and adjust to 20-40ng/μl. Make sure to make a fresh DNA solution. Note: in the event that the sample concentration is below the recommended range and a weak amplification occurs, sequence the sample. Acceptable sequencing and typing results may be achieved. Poor quality or degraded DNA Run genomic DNA on a 1% agarose gel to evaluate quality. Intact gdna should be ~3000 bp. V2, 2014/07 30 OF 40

31 Excessive background (baseline noise) ExoI/SAP not added to amplicons prior to sequencing. ExoI/SAP not thoroughly combined with the PCR product. No or poor clean-up of sequencing reactions performed Signal strength too high Poor or incorrect matrix Poor injection Injection time set too high Incorrect mobility file was chosen. Peaks will be shifted or will be on top of each other. Add ExoI/SAP to the PCR products for enzymatic clean-up. Be sure to vortex thoroughly after the addition of ExoI/SAP. Be sure to perform clean-up of sequencing products. When using sephadex, be sure to pipet the sequencing reaction on the middle of the sephadex column. See Excessive signal strength below. Repeat the spectral calibration and re-inject samples. Re-inject samples. Reduce injection time and re-inject. Choose correct mobility file. V2, 2014/07 31 OF 40

32 Weak signal Injection time needs to be increased. Excessive dye blobs No or poor clean-up of sequencing reactions performed Poor sequencing reaction due to error in pipetting or weak amplification product Excessive Signal Strength Injection time set too high Repeat sequencing reactions and increase injection time. Be sure to perform clean-up of sequencing reactions. When using Ethanol/EDTA precipitation, be sure to dry the pellets properly before reconstituting in HiDi formamide. When using sephadex, be sure to pipet the sequencing reaction on the middle of the sephadex column. Be sure that both the ExoI/SAP treated amplicon and the correct sequencing mix are added and combined. In the case of a weak amplification, confirm the intensity of the amplicon by running an agarose gel. Reduce injection time and re-inject. V2, 2014/07 32 OF 40

33 NOTES V2, 2014/07 33 OF 40

34 14 LIMITED LICENSE AGREEMENT Use of this product signifies the agreement of any purchaser or user of the GenDx SBTessenz kits with the following terms: The SBTessenz kits may be used solely in accordance with the SBTessenz IFU and for use with components contained in the kit only. GenDx 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 GenDx SBTessenz IFU and additional protocols available at Other than expressly stated licenses, GenDx 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 onetime use and may not be re-used, re-furbished, or re-sold. GenDx specifically disclaims any other licenses, expressed or implied other than those expressly stated. 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. GenDx may enforce the prohibitions of this Limited V2, 2014/07 34 OF 40

35 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 Trademarks: SBTessenz, SBTexcellerator and SBTengine are registered trade marks of Genome Diagnostics. QIAGEN : QIAGEN Group. ABI PRISM and BigDye : Applera Corporation. V2, 2014/07 35 OF 40

36 Edition 2, 2014/07 ORDERING INFORMATION GenDx products are supported either directly or by your local GenDx distributor or reseller. Please contact your local GenDx distributor ( or GenDx Customer Support team at or for any product information or quote request. GenDx Alexander Numan Building Yalelaan CM Utrecht The Netherlands Phone: +31 (0) Fax: +31 (0) www: GenDx, all rights reserved. V2, 2014/07 36 OF 40