SBTexcellerator HLA Handbook

Transcription

1 Third Edition July 2008 SBTexcellerator HLA Handbook For high-resolution HLA sequencing-based typing (SBT) 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. Licensed from Genome Diagnostics BV Sample & Assay Technologies

2 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

3 Contents Kit Contents 4 For HLA Class I 4 For HLA Class II 6 Shipping and Storage 7 Product Use Limitations 7 Product Warranty and Satisfaction Guarantee 8 Technical Assistance 8 Safety Information 8 Introduction 10 Principle and procedure 10 Equipment and Reagents to Be Supplied by User 12 Protocols 1: HLA Class I Locus Amplification 14 2: HLA-DRB1 Locus Amplification 17 3: HLA-DQB1 Locus Amplification 20 4: HLA-DPB1 Locus Amplification 23 5: Sequencing of HLA Loci 26 6: Cleanup and Analysis of HLA Sequencing Products 29 Troubleshooting Guide 31 References 35 Appendix A: Control of Contamination 35 Appendix B: Cleanup of HLA Sequencing Products Using the DyeEx 2.0 Spin Kit 36 Ordering Information 37 SBTexcellerator HLA Handbook 07/2008 3

4 Kit Contents For HLA Class I SBTexcellerator Core Kits HLA-A (50) HLA-B (50) HLA-C (50) Catalog no Number of reactions HLA-A Amp Forward and Reverse Primers (red cap) 1 tube HLA-A Sequencing Primers (yellow cap) HLA-B Amp Forward and Reverse Primers (red cap) HLA-B Sequencing Primers (yellow cap) HLA-C Amp Forward and Reverse Primers (red cap) HLA-C Sequencing Primers (yellow cap) Multiple tubes* 1 tube Multiple tubes* 1 tube Multiple tubes* RNase-Free Water (clear cap) 1 tube 1 tube 1 tube TE Buffer (clear cap) 1 tube 1 tube 1 tube HLA-A Protocol Sheet 1 HLA-B Protocol Sheet 1 HLA-C Protocol Sheet 1 Handbook * See the protocol sheet for primer details. 4 SBTexcellerator HLA Handbook 07/2008

5 SBTexcellerator Extended Kits HLA-A (50) HLA-B (50) HLA-C (50) Catalog no Number of reactions HLA-A GSSP Primers (green cap) Multiple tubes* HLA-B Sequencing Primers (yellow cap) Multiple tubes* HLA-B GSSP Primers (green cap) Multiple tubes* HLA-C Sequencing Primers (yellow cap) Multiple tubes* HLA-C GSSP Primers (green cap) Multiple tubes* RNase-Free Water (clear cap) 1 tube 1 tube 1 tube TE Buffer (clear cap) 1 tube 1 tube 1 tube HLA-A Protocol Sheet 1 HLA-B Protocol Sheet 1 HLA-C Protocol Sheet 1 * See the protocol sheet for primer details. SBTexcellerator HLA Handbook 07/2008 5

6 For HLA Class II SBTexcellerator Core Kits HLA-DPB1 (50) HLA-DQB1 (50) HLA-DRB1 (50) Catalog no Number of reactions HLA-DPB1 Amp Forward and Reverse Primers (red cap) 1 tube HLA-DPB1 Sequencing Primers (yellow cap) HLA-DQB1 Amp Forward and Reverse Primers (red cap) Multiple tubes* 1 tube HLA-DQB1 Sequencing Primers (yellow cap) Multiple tubes* HLA-DRB1 Amp Forward and Reverse Primers (red cap) HLA-DRB1 Sequencing Primers (yellow cap) HLA-DRB1 GSSP primers (green cap) RNase-Free Water (clear cap) 1 tube Multiple tubes* 1 tube* 1 tube 1 tube 1 tube TE Buffer (clear cap) 1 tube HLA-DPB1 Protocol Sheet 1 HLA-DQB1 Protocol Sheet 1 HLA-DRB1 Protocol Sheet 1 Handbook * See the protocol sheet for primer details. 6 SBTexcellerator HLA Handbook 07/2008

7 SBTexcellerator Extd. Kits HLA-DPB1 (50) HLA-DQB1 (50) HLA-DRB1 (50) Catalog no Number of reactions HLA-DPB1 GSSP Primers (green cap) Multiple tubes* HLA-DQB1 GSSP Primers (green cap) Multiple tubes* HLA-DRB1 Sequencing Primers (yellow cap) HLA-DRB1 GSSP Primers (green cap) RNase-Free Water (clear cap) 1 tube* Multiple tubes* 1 tube 1 tube 1 tube TE Buffer (clear cap) 1 tube HLA-DPB1 Protocol Sheet 1 HLA-DQB1 Protocol Sheet 1 HLA-DRB1 Protocol Sheet 1 * See the protocol sheet for primer details. Shipping and Storage SBTexcellerator HLA Kits are shipped at ambient temperature and should be stored at 15 to 30 C upon arrival. SBTexcellerator HLA Kits are stable until the kit expiration date when stored under these conditions. 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. The performance characteristics of the product have not been established, and it is not cleared or approved for clinical use by the United States FDA. In addition, the product is not CE marked for IVD use in the European Union. To ensure the best performance of SBTexcellerator HLA Kits, use the products with the materials, reagents, and equipments recommended in Equipment and SBTexcellerator HLA Handbook 07/2008 7

8 Reagents to Be Supplied by User, page 12. Use of other materials must be validated by user. 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 request, 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 ). 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 SBTexcellerator HLA Kits or QIAGEN products in general, please do not hesitate 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 or call one of the QIAGEN Technical Service Departments or local distributors (see back cover or visit ). 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 8 SBTexcellerator HLA Handbook 07/2008

9 convenient and compact PDF format at 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: SBTexcellerator HLA Handbook 07/2008 9

10 Introduction SBTexcellerator HLA Kits* are primer sets dedicated for high-resolution HLA sequencing-based typing (SBT). Principle and procedure HLA locus-specific amplification is performed in a thermal cycler using the amplification primer mix, template DNA, and the QIAGEN LongRange PCR Kit. Before sequencing, the PCR products are cleaned up using the QIAquick PCR Purification Kit (or alternative methods) to remove unincorporated primers and nucleotides. Sequencing is performed using BigDye Terminator sequencing chemistry. The final reactions are purified using DyeEx Kits (or alternative methods) to remove unincorporated sequencing primers and residual nucleotides. Denatured samples are loaded on an automated sequencing instrument. For high resolution typing of HLA Class I and Class II, sequencing of the exons of the HLA locus is required using the sequencing primers in the SBTexcellerator HLA Core Kits. Sequencing of other exons or the use of group-specific sequencing primers (GSSP), included in the SBTexcellerator HLA Extended Kits, is only required when ambiguities need to be resolved. * Licensed from Genome Diagnostics BV. 10 SBTexcellerator HLA Handbook 07/2008

11 Workflow of SBTexcellerator HLA procedure Amplify HLA Class I gene or specific exons of HLA Class II loci using forward and reverse primers and QIAGEN LongRange PCR Kit Confirm PCR products by agarose gel electrophoresis Clean up PCR products using QIAquick PCR Purification Kit (manual or fully automated on the QIAcube ) (or alternative) Perform sequencing reactions for individual exons using sequencing primers Clean up sequencing reactions using DyeEx Kits (or alternative) Perform analysis in SBTengine software Ambiguity present Sequence the additional regions or use GSSP(s), as indicated by the SBTengine software, to resolve ambiguities Allele assignment (unambiguous) Approve final analysis SBTexcellerator HLA Handbook 07/

12 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. For HLA locus amplification QIAGEN LongRange PCR Kit (cat. nos , , or ) Ice Pipets and pipet tips (use of pipet tips with hydrophobic filters is strongly recommended; see Appendix A, page 35) Thermal cycler Microcentrifuge Vortexer PCR tubes (use thin-walled 0.2 ml PCR tubes recommended by the manufacturer of your thermal cycler) Agarose gel electrophoresis system For sequencing of HLA loci QIAquick PCR Purification Kit (cat. nos or 28106);* alternatively, enzymatic cleanup methods can be used Pipets and pipet tips Thermal cycler Ethanol (96 100%) Microcentrifuge 1.5 or 2 ml microcentrifuge tubes 3 M sodium acetate, ph 5.0, may be necessary Optional: Distilled water or TE buffer (10 mm Tris Cl, 1 mm EDTA, ph 8) for elution of DNA. BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, cat. nos , , , or ) or BigDye Terminator v3.1 Cycle Sequencing Kit (cat. nos , , , , or )* * This is not a complete list of suppliers and does not include many important vendors of biological supplies. Do not use denaturated alcohol, which contains other substances such as methanol or methylethylketone. 12 SBTexcellerator HLA Handbook 07/2008

13 For cleanup and analysis of HLA sequencing products DyeEx 96 Kit (cat. nos or 63183) or DyeEx 2.0 Spin Kit (cat. nos or 63206);* alternatively, other size-exclusion methods or ethanol precipitation of sequencing products can be used Pipets and pipet tips Multichannel pipet (recommended to facilitate handling when working with the DyeEx 96 Kit) Using the DyeEx 96 Kit: QIAGEN Centrifuge 4-15C or 4K15C (see page 41 for ordering information) with Plate Rotor 2 x 96 (cat. no ) or equivalent centrifuge system (The use of DyeEx 96 plates requires a suitable centrifuge, rotor, and adapters. Rotor and adapters must be capable of centrifuging microplates of 4.5 cm total height.) Using the DyeEx 96 Kit: elution plate (96-Well Microplates RB, cat. no , or other 96-well plate or 12 x 8-well strips) Using the DyeEx 2.0 Spin Kit: Microcentrifuge Distilled water Capillary sequencer (e.g., ABI PRISM 3100 or Applied Biosystems 3130 Genetic Analyzers, or ABI PRISM 3700 or Applied Biosystems 3730 DNA Analyzers, Applied Biosystems)* SBTengine software (see page 39 for ordering information) or other suitable HLA typing software to analyze sequence files and to create HLA typing reports * This is not a complete list of suppliers and does not include many important vendors of biological supplies. SBTexcellerator HLA Kits have been tested with various polymers and sequencing chemistries. SBTexcellerator HLA Handbook 07/

14 Protocol 1: HLA Class I Locus Amplification This protocol is for amplification of HLA-A, HLA-B, or HLA-C genes using the QIAGEN LongRange PCR Kit and the respective SBTexcellerator HLA Kit. For amplification of HLA Class II loci, see Protocol 2: HLA-DRB1 Locus Amplification, page 17, Protocol 3: HLA-DQB1 Locus Amplification, page 20, and Protocol 4: HLA-DPB1 Locus Amplification, page 23. Important points before starting Purified DNA should have an A 260 /A 280 ratio between 1.7 and 1.9. We recommend using one of the following kits for purification of high-quality DNA from whole blood (see pages for ordering information). QIAamp DNA Blood Mini Kit EZ1 DNA Blood 200 μl Kit EZ1 DNA Blood 350 μl Kit Gentra Puregene Blood Kit If necessary, DNA should be diluted in RNase-free water before use. Blood samples should be collected in tubes with ACD* or EDTA* as an anticoagulant. Do not use heparinized samples. Read the QIAGEN LongRange PCR Handbook, QIAquick Spin Handbook, and DyeEx Handbook, paying particular attention to the Safety Information and Important Notes sections before beginning the procedure. Set up all reactions on ice. Things to do before starting Briefly centrifuge the tube containing the HLA Amp forward and reverse primers (red cap) before opening for the first time. Resuspend the primers in 55 μl RNase-free water or TE buffer (provided). * 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. 14 SBTexcellerator HLA Handbook 07/2008

15 Procedure 1. Thaw 10x LongRange PCR Buffer, dntp mix, RNase-free water, and primer solutions. Mix the solutions thoroughly and centrifuge briefly before use. 2. Prepare a reaction mix as shown in Table 1. Important: Set up all reactions on ice. The reaction mix typically contains all of the components needed for PCR except the template DNA. Prepare a volume of reaction mix at least 10% greater than that required for the total number of PCR assays to be performed. The optimal amount of template DNA to use in the reaction is 100 ng. However, template DNA in the range of ng (in 1 5 μl) can be used without affecting results. To streamline the process, validate your DNA purification procedure so that you can use a set volume corresponding to ng DNA. Table 1. Composition of reaction mix for HLA-A, HLA-B, and HLA-C locus amplification Component LongRange PCR Buffer with Mg 2+, 10x Volume in each reaction Final concentration 2.5 μl 1x; 2.5 mm Mg 2+ dntp mix (10 mm each) 1.25 μl 500 μm of each dntp HLA Amp Forward and Reverse Primers (red cap) 1 μl LongRange PCR Enzyme Mix 0.4 μl 2 units per reaction RNase-free water Template DNA Added at step 5 Total volume Variable ( μl) Variable (1 5 μl) 25 μl ng per reaction (optimal 100 ng) 3. Mix the reaction mix thoroughly, and centrifuge briefly. 4. Dispense the appropriate amount of the reaction mix into each PCR tube. The appropriate volume is 25 μl minus the amount of DNA added in step 5. SBTexcellerator HLA Handbook 07/

16 5. Add 1 5 μl template DNA ( ng) to each tube containing reaction mix. The final volume is 25 μl. The optimal amount of template DNA to use in the reaction is 100 ng. However, template DNA in the range of ng (in 1 5 μl) can be used without affecting results. 6. Program the thermal cycler according to the manufacturer s instructions, using the conditions outlined in Table 2. Table 2. Cycling protocol Comments Initial activation step: 3 min 95 C Initial denaturation of template DNA. 3-step cycling: Denaturation 15 s 93 C Do not exceed this temperature. Annealing 30 s 65 C Extension 3 min 68 C PCR products are approximately 3.5 kb. Number of cycles 35 Final extension: 10 min 68 C End of PCR cycling: Indefinite 4 C 7. Important: For a simplified hot start, place the tubes immediately into a thermal cycler that is preheated to 95 C and start the cycling program as outlined in Table 2. Use the simplified hot start to ensure PCR specificity. After amplification, samples can be stored overnight at 2 8 C. Cleanup of the PCR products (page 26, steps 1 10) should be carried out within 24 h. 8. Confirm the PCR products using an appropriate detection system such as agarose gel electrophoresis. Prepare a 1% w/v agarose gel according to your laboratory protocol, and analyze 2 μl of each PCR assay. See Table 2 for approximate size of PCR products. 9. Proceed with Protocol 5: Sequencing of HLA Loci, page SBTexcellerator HLA Handbook 07/2008

17 Protocol 2: HLA-DRB1 Locus Amplification This protocol is for amplification of part of the HLA-DRB1 gene using the QIAGEN LongRange PCR Kit and the SBTexcellerator HLA-DRB1 Kit. For amplification of HLA-DQB1 and HLA-DPB1, see Protocol 3: HLA-DQB1 Locus Amplification, page 20 and Protocol 4: HLA-DPB1 Locus Amplification, page 23. For amplification of HLA Class I loci, see Protocol 1: HLA Class I Locus Amplification, page 14. Important points before starting Purified DNA should have an A 260 /A 280 ratio between 1.7 and 1.9. We recommend using one of the following kits for purification of high-quality DNA from whole blood (see pages for ordering information). QIAamp DNA Blood Mini Kit EZ1 DNA Blood 200 μl Kit EZ1 DNA Blood 350 μl Kit Gentra Puregene Blood Kit If necessary, DNA should be diluted in RNase-free water before use. Blood samples should be collected in tubes with ACD* or EDTA* as an anticoagulant. Do not use heparinized samples. Read the QIAGEN LongRange PCR Handbook, QIAquick Spin Handbook, and DyeEx Handbook, paying particular attention to the Safety Information and Important Notes sections before beginning the procedure. Set up all reactions on ice. Things to do before starting Briefly centrifuge the tube containing the HLA-DRB1 Amp forward and reverse primers (red cap) before opening for the first time. Resuspend the primers in 55 μl RNase-free water (provided). * 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. SBTexcellerator HLA Handbook 07/

18 Procedure 1. Thaw 10x LongRange PCR Buffer, dntp mix, RNase-free water, and primer solutions. Mix the solutions thoroughly and centrifuge briefly before use. 2. Prepare a reaction mix as shown in Table 3. Important: Set up all reactions on ice. The reaction mix typically contains all of the components needed for PCR except the template DNA. Prepare a volume of reaction mix at least 10% greater than that required for the total number of PCR assays to be performed. The optimal amount of template DNA to use in the reaction is 100 ng. However, template DNA in the range of ng (in 1 5 μl) can be used without affecting results. To streamline the process, validate your DNA purification procedure so that you can use a set volume corresponding to ng DNA. Table 3. Composition of reaction mix for HLA-DRB1 locus amplification Component LongRange PCR Buffer with Mg 2+, 10x Volume in each reaction Final concentration 2.5 μl 1x; 2.5 mm Mg 2+ dntp mix (10 mm each) 1.25 μl 500 μm of each dntp HLA-DRB1 Amp Forward and Reverse Primers (red cap) 1 μl LongRange PCR Enzyme Mix 0.4 μl 2 units per reaction RNase-free water Template DNA Added at step 5 Total volume Variable ( μl) Variable (1 5 μl) 25 μl ng per reaction (optimal 100 ng) 3. Mix the reaction mix thoroughly, and centrifuge briefly. 4. Dispense the appropriate amount of the reaction mix into each PCR tube. The appropriate volume is 25 μl minus the amount of DNA added in step SBTexcellerator HLA Handbook 07/2008

19 5. Add 1 5 μl template DNA ( ng) to each tube containing reaction mix. The final volume is 25 μl. The optimal amount of template DNA to use in the reaction is 100 ng. However, template DNA in the range of ng (in 1 5 μl) can be used without affecting results. 6. Program the thermal cycler according to the manufacturer s instructions, using the conditions outlined in Table 4. Table 4. Cycling protocol for HLA-DRB1 locus amplification Comments Initial activation step: 3 min 95 C Initial denaturation of template DNA. 3-step cycling: Denaturation 15 s 93 C Do not exceed this temperature. Annealing 30 s 65 C Extension 5 min 68 C PCR product is approximately 4.5 kb. Number of cycles 35 Final extension: 10 min 68 C End of PCR cycling: Indefinite 4 C 7. Important: For a simplified hot start, place the tubes immediately into a thermal cycler that is preheated to 95 C and start the cycling program as outlined in Table 4. Use the simplified hot start to ensure PCR specificity. After amplification, samples can be stored overnight at 2 8 C. Cleanup of the PCR products (page 26, steps 1 10) should be carried out within 24 h. 8. Confirm the PCR products using an appropriate detection system such as agarose gel electrophoresis. Prepare a 1% w/v agarose gel according to your laboratory protocol, and analyze 2 μl of each PCR assay. See Table 4 for approximate size of PCR products. 9. Proceed with Protocol 5: Sequencing of HLA Loci, page 26. SBTexcellerator HLA Handbook 07/

20 Protocol 3: HLA-DQB1 Locus Amplification This protocol is for amplification of part of the HLA-DQB1 gene using the QIAGEN LongRange PCR Kit and the SBTexcellerator HLA-DQB1 Kit. For amplification of HLA-DRB1 and HLA-DPB1, see Protocol 2: HLA-DRB1 Locus Amplification, page 17, and Protocol 4: HLA-DPB1 Locus Amplification, page 23. For amplification of HLA Class I loci, see Protocol 1: HLA Class I Locus Amplification, page 14. Important points before starting Purified DNA should have an A 260 /A 280 ratio between 1.7 and 1.9. We recommend using one of the following kits for purification of high-quality DNA from whole blood (see pages for ordering information). QIAamp DNA Blood Mini Kit EZ1 DNA Blood 200 μl Kit EZ1 DNA Blood 350 μl Kit Gentra Puregene Blood Kit If necessary, DNA should be diluted in RNase-free water before use. Blood samples should be collected in tubes with ACD* or EDTA* as an anticoagulant. Do not use heparinized samples. Read the QIAGEN LongRange PCR Handbook, QIAquick Spin Handbook, and DyeEx Handbook, paying particular attention to the Safety Information and Important Notes sections before beginning the procedure. Set up all reactions on ice. Things to do before starting Briefly centrifuge the tube containing the HLA-DQB1 Amp forward and reverse primers (red cap) before opening for the first time. Resuspend the primers in 55 μl RNase-free water (provided). * 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. 20 SBTexcellerator HLA Handbook 07/2008

21 Procedure 1. Thaw 10x LongRange PCR Buffer, dntp mix, RNase-free water, and primer solutions. Mix the solutions thoroughly and centrifuge briefly before use. 2. Prepare a reaction mix as shown in Table 5. Important: Set up all reactions on ice. The reaction mix typically contains all of the components needed for PCR except the template DNA. Prepare a volume of reaction mix at least 10% greater than that required for the total number of PCR assays to be performed. The optimal amount of template DNA to use in the reaction is 100 ng. However, template DNA in the range of ng (in 1 5 μl) can be used without affecting results. To streamline the process, validate your DNA purification procedure so that you can use a set volume corresponding to ng DNA. Table 5. Composition of reaction mix for HLA-DQB1 locus amplification Component LongRange PCR Buffer with Mg 2+, 10x Volume in each reaction Final concentration 2.5 μl 1x; 2.5 mm Mg 2+ dntp mix (10 mm each) 1.25 μl 500 μm of each dntp HLA-DQB1 Amp Forward and Reverse Primers (red cap) 1 μl LongRange PCR Enzyme Mix 0.4 μl 2 units per reaction Q-Solution, 5x 5 μl 1x RNase-free water Template DNA Added at step 5 Total volume Variable ( μl) Variable (1 5 μl) 25 μl ng per reaction (optimal 100 ng) 3. Mix the reaction mix thoroughly, and centrifuge briefly. 4. Dispense the appropriate amount of the reaction mix into each PCR tube. The appropriate volume is 25 μl minus the amount of DNA added in step 5. SBTexcellerator HLA Handbook 07/

22 5. Add 1 5 μl template DNA ( ng) to each tube containing reaction mix. The final volume is 25 μl. The optimal amount of template DNA to use in the reaction is 100 ng. However, template DNA in the range of ng (in 1 5 μl) can be used without affecting results. 6. Program the thermal cycler according to the manufacturer s instructions, using the conditions outlined in Table 6. Table 6. Cycling protocol for HLA-DQB1 locus amplification Comments Initial activation step: 3 min 95 C Initial denaturation of template DNA. 3-step cycling: Denaturation 15 s 93 C Do not exceed this temperature. Annealing 30 s 65 C Extension 4 min 68 C PCR product is approximately 4 kb. Number of cycles 35 Final extension: 10 min 68 C End of PCR cycling: Indefinite 4 C 7. Important: For a simplified hot start, place the tubes immediately into a thermal cycler that is preheated to 95 C and start the cycling program as outlined in Table 6. Use the simplified hot start to ensure PCR specificity. After amplification, samples can be stored overnight at 2 8 C. Cleanup of the PCR products (page 26, steps 1 10) should be carried out within 24 h. 8. Confirm the PCR products using an appropriate detection system such as agarose gel electrophoresis. Prepare a 1% w/v agarose gel according to your laboratory protocol, and analyze 2 μl of each PCR assay. See Table 6 for approximate size of PCR products. 9. Proceed with Protocol 5: Sequencing of HLA Loci, page SBTexcellerator HLA Handbook 07/2008

23 Protocol 4: HLA-DPB1 Locus Amplification This protocol is for amplification of part of the HLA-DPB1 gene using the QIAGEN LongRange PCR Kit and the SBTexcellerator HLA-DPB1 Kit. For amplification of HLA-DRB1 and HLA-DQB1, see Protocol 2: HLA-DRB1 Locus Amplification, page 17, and Protocol 3: HLA-DQB1 Locus Amplification, page 20. For amplification of HLA Class I loci, see Protocol 1: HLA Class I Locus Amplification, page 14. Important points before starting Purified DNA should have an A 260 /A 280 ratio between 1.7 and 1.9. We recommend using one of the following kits for purification of high-quality DNA from whole blood (see pages for ordering information). QIAamp DNA Blood Mini Kit EZ1 DNA Blood 200 μl Kit EZ1 DNA Blood 350 μl Kit Gentra Puregene Blood Kit If necessary, DNA should be diluted in RNase-free water before use. Blood samples should be collected in tubes with ACD* or EDTA* as an anticoagulant. Do not use heparinized samples. Read the QIAGEN LongRange PCR Handbook, QIAquick Spin Handbook, and DyeEx Handbook, paying particular attention to the Safety Information and Important Notes sections before beginning the procedure. Set up all reactions on ice. Things to do before starting Briefly centrifuge the tube containing the HLA-DPB1 Amp forward and reverse primers (red cap) before opening for the first time. Resuspend the primers in 55 μl RNase-free water or TE buffer (provided). * 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. SBTexcellerator HLA Handbook 07/

24 Procedure 1. Thaw 10x LongRange PCR Buffer, dntp mix, RNase-free water, and primer solutions. Mix the solutions thoroughly and centrifuge briefly before use. 2. Prepare a reaction mix as shown in Table 7. Important: Set up all reactions on ice. The reaction mix typically contains all of the components needed for PCR except the template DNA. Prepare a volume of reaction mix at least 10% greater than that required for the total number of PCR assays to be performed. The optimal amount of template DNA to use in the reaction is 100 ng. However, template DNA in the range of ng (in 1 5 μl) can be used without affecting results. To streamline the process, validate your DNA purification procedure so that you can use a set volume corresponding to ng DNA. Table 7. Composition of reaction mix for HLA-DPB1 locus amplification Component LongRange PCR Buffer with Mg 2+, 10x Volume in each reaction Final concentration 2.5 μl 1x; 2.5 mm Mg 2+ dntp mix (10 mm each) 1.25 μl 500 μm of each dntp HLA-DPB1 Amp Forward and Reverse Primers (red cap) 1 μl LongRange PCR Enzyme Mix 0.4 μl 2 units per reaction RNase-free water Template DNA Added at step 5 Total volume Variable ( μl) Variable (1 5 μl) 25 μl ng per reaction (optimal 100 ng) 3. Mix the reaction mix thoroughly, and centrifuge briefly. 4. Dispense the appropriate amount of the reaction mix into each PCR tube. The appropriate volume is 25 μl minus the amount of DNA added in step SBTexcellerator HLA Handbook 07/2008

25 5. Add 1 5 μl template DNA ( ng) to each tube containing reaction mix. The final volume is 25 μl. The optimal amount of template DNA to use in the reaction is 100 ng. However, template DNA in the range of ng (in 1 5 μl) can be used without affecting results. 6. Program the thermal cycler according to the manufacturer s instructions, using the conditions outlined in Table 8. Table 8. Cycling protocol for HLA-DPB1 locus amplification Comments Initial activation step: 3 min 95 C Initial denaturation of template DNA. 3-step cycling: Denaturation 15 s 93 C Do not exceed this temperature. Annealing 30 s 65 C Extension 30 s 68 C PCR product is 320 bp. Number of cycles 35 Final extension: 10 min 68 C End of PCR cycling: Indefinite 4 C 7. Important: For a simplified hot start, place the tubes immediately into a thermal cycler that is preheated to 95 C and start the cycling program as outlined in Table 8. Use the simplified hot start to ensure PCR specificity. After amplification, samples can be stored overnight at 2 8 C. Cleanup of the PCR products (page 26, steps 1 10) should be carried out within 24 h. 8. Confirm the PCR products using an appropriate detection system such as agarose gel electrophoresis. Prepare a 1% w/v agarose gel according to your laboratory protocol, and analyze 2 μl of each PCR assay. See Table 8 for approximate size of PCR products. 9. Proceed with Protocol 5: Sequencing of HLA Loci, page 26. SBTexcellerator HLA Handbook 07/

26 Protocol 5: Sequencing of HLA Loci This procedure is optimized for BigDye Terminator chemistry, with subsequent analysis on ABI PRISM 3100 or Applied Biosystems 3130 Genetic Analyzers, or ABI PRISM 3700 or Applied Biosystems 3730 DNA Analyzers. Important points before starting Read the QIAquick Spin Handbook and the BigDye Terminator protocol manual, paying particular attention to the Safety Information and Important Notes sections before beginning the procedure. As an alternative to PCR cleanup using the QIAquick PCR Purification Kit, enzymatic cleanup methods can also be used. The yellow color of Buffer PBI in the QIAquick PCR Purification Kit indicates a ph 7.5. All centrifugation steps are carried out at 10,000 x g in a conventional tabletop microcentrifuge at room temperature (15 25 C). Things to do before starting Add ethanol (96 100%) to Buffer PE in the QIAquick PCR Purification Kit before use (see bottle label for volume). Briefly centrifuge the tubes containing the sequencing primers (yellow and green caps) before opening for the first time. Resuspend each primer in 55 μl RNase-free water or TE buffer (provided). Procedure 1. Add 115 μl Buffer PBI to the PCR sample (23 μl) and mix. 2. Check that the color of the mixture is yellow (similar to Buffer PBI without the PCR sample). If the color of the mixture is orange or violet, add 10 μl of 3 M sodium acetate, ph 5.0, and mix. The color of the mixture will turn to yellow. 3. Place a QIAquick spin column in a 2 ml collection tube (provided). 4. To bind DNA, apply the sample to the QIAquick column and centrifuge for s. 5. Discard flow-through. Place the QIAquick column back into the same tube. Collection tubes are re-used to reduce plastic waste. 6. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for s. 7. Discard flow-through and repeat step 6 once. 26 SBTexcellerator HLA Handbook 07/2008

27 8. Discard flow-through and place the QIAquick column back in the same tube. Centrifuge the column for an additional 1 min. Important: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation. 9. Place QIAquick column in a clean 1.5 ml microcentrifuge tube. 10. To elute DNA, add 50 μl Buffer EB (10 mm Tris Cl, ph 8.5) or water (ph ) to the center of the QIAquick membrane. Let the column stand for 1 min, and centrifuge the column for 1 min. Important: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is approximately 40 μl from 50 μl elution buffer. Elution efficiency is dependent on ph. The maximum elution efficiency is achieved between ph 7.0 and 8.5. When using water, make sure that the ph value is within this range, and store DNA at 20 C as DNA may degrade in the absence of a buffering agent. 11. Prepare a sequencing master mix according to Table 9. The sequencing master mix should contain all of the components needed for all sequence tests to be performed. Prepare a volume of sequencing master mix at least 10% greater than that required for the total number of sequencing assays to be performed. Table 9. Composition of sequencing master mix for HLA locus sequencing Volume in each Final Component reaction concentration BDT Ready Reaction Premix, 2.5x 2 μl 1x BDT Buffer, 5x 1 μl 1x RNase-free water 5 μl Sequencing primer (yellow or green cap) Total 1 μl 9 μl 12. Gently mix the sequencing master mix and centrifuge briefly. 13. Dispense 9 μl of the sequencing master mix into each PCR tube. 14. Add 1 μl purified PCR product (from step 10) to each tube containing sequencing master mix. The final volume is 10 μl. SBTexcellerator HLA Handbook 07/

28 15. Program the thermal cycler according to the manufacturer s instructions, using the conditions outlined in Table 10. Table 10. Cycling protocol Comments Initial denaturation: 10 s 96 C Initial denaturation of template DNA. 3-step cycling: Denaturation 10 s 96 C Annealing 10 s 50 C Extension 4 min 60 C Number of cycles 30 End of PCR cycling: Indefinite 4 C 16. Place the tubes immediately into a thermal cycler and start the cycling program as outlined in Table After the sequencing reaction is finished, proceed with Protocol 6: Cleanup and Analysis of HLA Sequencing Products, page SBTexcellerator HLA Handbook 07/2008

29 Protocol 6: Cleanup and Analysis of HLA Sequencing Products This procedure uses the DyeEx 96 Kit for cleanup of HLA sequencing products. For cleanup using the DyeEx 2.0 Spin Kit, see Appendix B, page 36. Alternatively, other equivalent cleanup methods can be used. Important points before starting Read the DyeEx Handbook, paying particular attention to the Safety Information and Important Notes sections before beginning the procedure. As an alternative to cleanup using DyeEx Kits, other equivalent sizeexclusion methods or ethanol precipitation of sequencing products can also be used. The use of DyeEx 96 plates requires a suitable centrifuge, rotor, and adapters. Rotor and adapters must be capable of centrifuging microplates of 4.5 cm total height, such as the Plate Rotor 2 x 96 (see page 41 for ordering information). DyeEx 96 plates must be centrifuged at 1000 x g. After centrifugation, the gel-bed surface in the wells of the DyeEx 96 plate may vary due to the differing centrifugal force in the different wells. This is normal and has no effect on the performance of the DyeEx 96 procedure. Always use the waste collection plates provided with the DyeEx 96 Kit. We do not recommend any other waste collection plates for use with the DyeEx 96 Kit. Procedure 1. Take the DyeEx 96 plate out of the bag, and remove the tape sheets from the top and bottom of the DyeEx 96 plate. When handling the DyeEx 96 plate, ensure that it remains horizontal. It is easier to remove the tape from the bottom first. 2. Place the DyeEx 96 plate on the top of the collection plate (provided), and centrifuge for 1 min at 1000 x g. The collection plates are reusable. Discard the flow-through. Note: Always use the waste collection plates provided with the DyeEx 96 Kit. We do not recommend any other collection plates for use with the DyeEx 96 Kit. 3. Place the DyeEx 96 plate on top of the collection plate. Add 300 μl water to each well, and centrifuge for 3 min at 1000 x g. We recommend the use of deionized water. SBTexcellerator HLA Handbook 07/

30 4. Carefully place the DyeEx 96 plate on an appropriate elution plate with a suitable adapter. Appropriate elution plates include 96-well microplate, cat. no , or other 96-well plates or 12 x 8-well strips. Note: To ensure that the DyeEx 96 plate sits securely in the centrifuge rotor, the tops of the wells of the elution plate should be in direct contact with the base of the DyeEx 96 plate. 5. Slowly apply the sequencing raction (10 μl) to the gel bed of each well. Note: Pipet the sequencing reaction directly onto the center of the gel-bed surface (see DyeEx Handbook). Do not allow the reaction mixture or the pipet tip to touch the sides of the wells. The samples should be pipetted slowly so that they are absorbed into the gel and do not flow down the sides of the gel bed. Avoid touching the gel-bed surface with the pipet tip. 6. Centrifuge for 3 min at 1000 x g. The eluate contains the purified DNA. 7. Load the samples into the sequencer. Run the sequencer according to the manufacturer s instructions. Note: Matrix standards must be run for first-time use of BigDye Terminator reactions. Follow the instructions provided in the BigDye Terminator Cycle Sequencing Kit or by the manufacturer. SBTexcellerator HLA Kits have been tested with various polymers and sequencing chemistries. 8. Analyze HLA data. Use sequencing analysis software to process collected raw data and SBTengine software or other suitable HLA typing software to analyze the sequence files and to create HLA typing reports. 30 SBTexcellerator HLA Handbook 07/2008

31 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: 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 ). Little or no PCR product a) LongRange PCR Enzyme Mix was not added to the amplification mix or not mixed properly when added Comments and suggestions Repeat amplification paying attention to the addition and mixing of LongRange PCR Enzyme Mix with the amplification mix. b) Cycling conditions not optimal c) DNA concentration not optimal d) Poor-quality or degraded genomic DNA If using a fast thermal cycler, reduce the ramp rate to 1 C/s. If using a Biometra thermal cycler, raise the denaturation temperature from 93 C to 95 C in the 3-step cycling protocol for HLA locus amplification. Requantify the DNA and adjust to 50 ng/μl. If the sample concentration is below the recommended range and little or no amplification product is visible, sequence the sample. Acceptable sequence and typing may be achievable. Run genomic DNA on a 1% agarose gel to evaluate quality. Purified DNA should have an A 260 /A 280 ratio between 1.7 and 1.9. For best DNA quality, we recommend using one of the DNA purification kits listed on pages 14, 17, 20, or 23. SBTexcellerator HLA Handbook 07/

32 Unusual PCR products a) Two PCR products visible after amplification of HLA- DRB1 or HLA-DQB1 locus b) HLA-DRB1 or HLA-DQB1 amplicons of different samples have different sizes Excessive background noise a) PCR products not cleaned up prior to sequencing b) No or poor cleanup of sequencing reactions Comments and suggestions In a heterozygous sample, two bands may appear for the HLA-DRB1 or HLA-DQB1 PCR products due to length polymorphism in intronic regions of the HLA-DRB1 or HLA- DQB1 gene. Due to length polymorphisms in intronic regions of the HLA-DRB1 or HLA-DQB1 gene, amplicons may vary in size in different samples. Clean up the PCR products using the QIAquick PCR Purification Kit before using them in the sequencing reaction. Be sure to perform cleanup of sequencing reactions using the DyeEx 2.0 Spin Kit or the DyeEx 96 Kit. Pipet the sample directly onto the center of the gel-bed surface (see DyeEx Handbook). Do not allow the reaction mixture or the pipet tip to contact the sides of the gelbed or the sides of the DyeEx 2.0 spin columns or wells of the DyeEx plates. c) Signal strength too high See Excessive signal strength below. d) Poor or incorrect matrix Repeat the spectral calibration and reinject samples. e) Poor injection Reinject samples f) Injection time set too high Reduce injection time and reinject. Signal strengths of are optimal. Samples of poor quality may have lower signal strengths but may still be analyzed and typed. Some samples may have signals that are over 1500 and will not have excess background. g) Peaks shifted or on top of each other Incorrect mobility file chosen. Choose correct mobility file. 32 SBTexcellerator HLA Handbook 07/2008

33 h) Poor sequence quality in one of the HLA-DRB1 exon 2 forward sequences Weak signal a) Injection time needs to be increased b) PCR product concentration too low Comments and suggestions There are 2 forward sequencing primers for exon 2. Both should be used routinely. Primer R3 is specific for sequencing alleles in group DR1/2/3/5/6/8/10. Primer R4 is specific for sequencing alleles present in group DR4/7/9. With homozygous samples, sequences will be obtained from only one of the primers. In heterozygous samples possessing 2 alleles from the same set of groups (e.g., group DR1 and group DR6), heterozygous sequence data is to be expected from the primer for the relevant groups, and the other primer will generate bad or no sequence. The SBTengine software will reject this sequence data, in most cases, so it does not interfere with typing analysis. Repeat sequencing reactions and increase injection time. Increase the amount of PCR product in the sequencing reaction, and reduce the amount of RNase-free water proportionately. Excessive dye blobs a) No or poor cleanup of sequencing reactions b) Poor sequencing reaction due to error in pipetting or weak amplification product Be sure to perform cleanup of sequencing reactions using the DyeEx 2.0 Spin Kit or the DyeEx 96 Kit. Pipet the sample directly onto the center of the gel-bed surface (see DyeEx Handbook). Do not allow the reaction mixture or the pipet tip to contact the sides of the gelbed or the sides of the DyeEx 2.0 spin columns or wells of the DyeEx plates. Be sure that both the cleaned-up amplicon and the correct sequencing mix are added and combined. In the case of weak amplification, confirm the intensity of the amplicon by running an agarose gel. SBTexcellerator HLA Handbook 07/

34 Excessive signal strength Comments and suggestions a) PCR product too concentrated b) Too much BDT Ready Reaction Premix in the sequencing reaction Dilute the PCR product with RNase-free water before sequencing. Reduce the amount of BDT Reaction Premix and adjust the amount of BDT Buffer according to the manufacturer s instructions. c) Injection time set too high Reduce injection time and reinject. Signal strengths of are optimal. Samples of poor quality may have lower signal strengths but may still be analyzed and typed. Some samples may have signals that are over 1500 and will not have excess background. 34 SBTexcellerator HLA Handbook 07/2008

35 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 or contact QIAGEN Technical Services or your local distributor. Appendix A: Control of Contamination 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 pipets for the PCR amplification mix. Use of pipet tips with hydrophobic filters is strongly recommended. Prepare and freeze small aliquots of primer solutions and dntp mix. Use of fresh RNase-free water is strongly recommended. In case of contamination, laboratory benches, apparatus, and pipets can be decontaminated by cleaning them with a 1/10 dilution of a commercial bleach solution.* Afterwards, the benches and pipets should be rinsed with RNase-free water. 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 preventing amplification of contaminating DNA is to treat individual reaction mixtures with DNase I or restriction enzymes that cut between the binding sites of the amplification primers used, before adding the template DNA sample. * Most commercial bleach solutions are approximately 5.25% sodium hypochlorate. Sodium hypochlorate is an irritant and should be handled with caution. 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. SBTexcellerator HLA Handbook 07/

36 Appendix B: Cleanup of HLA Sequencing Products Using the DyeEx 2.0 Spin Kit Important points before starting Read the DyeEx Handbook, paying particular attention to the Safety Information and Important Notes sections before beginning the procedure. All centrifugation steps are performed at 750 x g in a conventional microcentrifuge. Procedure B1. Gently vortex the DyeEx spin column to resuspend the resin. B2. Loosen the cap of the spin column a quarter turn. This is necessary to avoid a vacuum inside the spin column. B3. Snap off the bottom closure of the DyeEx spin column, and place it in a 2 ml collection tube (provided). B4. Centrifuge for 3 min at 750 x g. B5. Add 300 μl water, and centrifuge for 3 min at 750 x g. We recommend the use of deionized water. B6. Carefully transfer the spin column to a clean microcentrifuge tube. Slowly apply the sequencing reaction (10 μl) to the gel bed. Note: Pipet the sequencing reaction directly onto the center of the slanted gel-bed surface (see DyeEx Handbook). Do not allow the reaction mixture or the pipet tip to touch the sides of the column. The sample should be pipetted slowly so that the drops are absorbed into the gel and do not flow down the sides of the gel bed. Avoid touching the gel-bed surface with the pipet tip. It is not necessary to replace the lid on the column. B7. Centrifuge for 3 min at 750 x g. B9. Remove the spin column from the microcentrifuge tube. The eluate contains the purified DNA. B10. Continue with step 7 of Protocol 6: Cleanup and Analysis of HLA Sequencing Products, page SBTexcellerator HLA Handbook 07/2008

37 Ordering Information SBTexcellerator HLA Kits and SBTengine Software are available from QIAGEN HLA distributors. USA and Canada Orders Fax Technical 800-DNA-PREP ( ) Rest of world Orders Fax Technical Other QIAGEN products are available from QIAGEN subsidiaries and distributors (see back cover or visit ). Product Contents Cat. no. SBTexcellerator HLA Kits for HLA Class I SBTexcellerator HLA-A Core Kit (50) For 50 HLA typings: HLA-A Amplification Primers, HLA-A Sequencing Primers, RNase-Free Water, and TE Buffer SBTexcellerator HLA-A Extended Kit (50) SBTexcellerator HLA-A Full Kit (50) SBTexcellerator HLA-B Core Kit (50) SBTexcellerator HLA-B Extended Kit (50) SBTexcellerator HLA-B Full Kit (50) For 50 HLA typings: HLA-A Group- Specific Sequencing Primers, RNase- Free Water, and TE Buffer; to be used with the SBTexcellerator HLA-A Core Kit For 50 HLA typings: SBTexcellerator HLA-A Core Kit and SBTexcellerator HLA-A Extended Kit For 50 HLA typings: HLA-B Amplification Primers, HLA-B Sequencing Primers, RNase-Free Water, and TE Buffer For 50 HLA typings: HLA-B Amplification Primers, HLA-B Sequencing Primers, HLA-B Group- Specific Sequencing Primers, RNase- Free Water, and TE Buffer; to be used with the SBTexcellerator HLA-B Core Kit For 50 HLA typings: SBTexcellerator HLA-B Core Kit and SBTexcellerator HLA-B Extended Kit SBTexcellerator HLA Handbook 07/