Instructions for Use PCR Amplification and Sequencing of the DRB3/DRB4/DRB5 Version No: 1.0 Issue Date: February 2012 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. Conexio Genomics Pty Ltd 8/31 Pakenham St Fremantle 6160 Western Australia Australia Page 1 of 15 For Research Use Only Contents PRINCIPLE ............................................................................................................................................ 3 KIT COMPOSITION ............................................................................................................................ 4 STORAGE REQUIREMENTS ............................................................................................................. 6 MATERIALS, REAGENTS AND EQUIPMENT NOT SUPPLIED ................................................. 6 SAMPLE REQUIREMENTS ................................................................................................................ 7 WARNINGS AND SAFETY PRECAUTIONS ................................................................................... 7 PROCEDURE ........................................................................................................................................... 8 1. 2. 3. 4. 5. 6. 7. PCR ............................................................................................................................................ 8 AGAROSE GEL ELECTROPHORESIS .............................................................................................. 9 PURIFICATION OF POSITIVE DRB3/DRB4/DRB5 PCR PRODUCT ............................................... 9 SEQUENCING REACTION ........................................................................................................... 10 PURIFICATION OF SEQUENCING REACTION PRODUCTS.............................................................. 11 DENATURATION & ELECTROPHORESIS OF SEQUENCING REACTION PRODUCTS ........................ 11 EDITING AND ANALYSIS OF ELECTROPHEROGRAMS .................................................................. 12 LIMITATIONS AND CAUTIONS ..................................................................................................... 12 LICENSE .............................................................................................................................................. 13 TROUBLESHOOTING ....................................................................................................................... 13 SUPPORT AND CONTACT DETAILS............................................................................................. 15 Page 2 of 15 For Research Use Only Principle The HLA Sequence Based Typing (SBT) procedure described here involves the locus specific PCR amplification of the HLA-DRB3/DRB4/DRB5 target amplicons which are visualised by agarose gel electrophoresis. Samples that are positive for HLADRB3/DRB4/DRB5 will produce two amplicons (the target amplicon and internal control) while negative samples will produce a single amplicon only (internal control). Positive samples are then further characterised by DNA sequencing following treatment with ExoSAP-IT® to remove unincorporated primers and dNTPs. The target amplicon is used as a template for direct automated fluorescent DNA sequencing using customized sequencing primers and the BigDye® Terminator sequencing chemistry available from Applied Biosystems™ by Life Technologies™. The extension products are purified according to the ethanol precipitation method and denatured using Hi-Di™ formamide available from Applied Biosystems™ by Life Technologies™, before separation and detection on an automated fluorescent DNA sequencer. It is recommended that the resulting data is analysed with Assign™ sequence analysis software from Conexio Genomics Pty Ltd. Page 3 of 15 For Research Use Only Kit Composition Kit PRE-PCR Contents† (No of vials) Catalogue No POST-PCR Contents (No of vials) Class II HLA-DRB3 AN-PD11.0(20) AN-PD11.0(50) HLA-DRB4 AN-PD12.0(20) AN-PD12.0(50) 20 tests 50 tests 20 tests 50 tests DNA POL – DRB3 1 x 9L HLA-DRB3 MIX 1 x 370L DNA POL – DRB3 1 x 18L HLA-DRB3 MIX 1 x 920L DNA POL – DRB4 1 x 9L HLA-DRB4 MIX 1 x 370L DNA POL – DRB4 1 x 18L HLA-DRB4 MIX 1 x 920L Page 4 of 15 DRB3EX2F DRB3EX2R 1 x 44L each DRB3EX2F DRB3EX2R 1 x 110L each DRB4EX2F DRB4EX2R 1 x 44L each DRB4EX2F DRB4EX2R 1 x 110L each For Research Use Only HLA-DRB5 AN-PD13.0(20) AN-PD13.0(50) 20 tests 50 tests DNA POL – DRB5 1 x 9L HLA-DRB5 MIX 1 x 370L DNA POL – DRB5 1 x 18L HLA-DRB5 MIX 1 x 920L † DRB5EX2F The PRE-PCR component of each kit consists of a vial/s of a locus-specific PCR mix (e.g. dNTPs, MgCl2, locus specific PCR primers, and a single vial of DNA polymerase (e.g. The POST-PCR kit contains sequencing primers (e.g. DRB3EX2F DRB5EX2R 1 x 44uL each DRB5EX3F DRB5EX2F DPB5EX2R 1 x 110uL each DRB5EX3F ) consisting of PCR buffer, HLA-DRB3 MIX DNA POL – DRB3 ). ). Page 5 of 15 For Research Use Only Storage Requirements The PRE- and POST-PCR boxes may be separated and stored in designated PRE- and POSTPCR freezers. When stored at -20C, the kit components can be used until the expiry date indicated on the outer kit containers. Materials, Reagents and Equipment Not Supplied PCR 1. Sterile water 2. Electronic or mechanical pipettes and aerosol-resistant tips 3. Thermal cycler with heated lid These kits have been tested using the following thermal cyclers: MJ Research PTC 225 DNA Engine DYAD™, Applied Biosystems™ by Life Technologies™ GeneAmp® PCR System 9700, and Eppendorf Mastercycler® Pro. Use of other thermal cyclers with these kits requires validation by the user. 4. 0.2mL thin-walled thermal cycling reaction tubes (8 well strips or 96 well plates). Use those recommended for use with your thermal cycler. 5. Sterile 1.5mL tubes 6. Sterile biological safety cabinet or hood. 7. Table top centrifuge with plate adapters and capacity to reach 2500 x g 8. Vortex 9. Agarose gel electrophoresis apparatus 10. 1% agarose (molecular biology grade) TBE gel containing 0.1g/mL ethidium bromide. 11. Loading buffer 12. PCR Marker suitable to cover range of 300 – 1300 bp 13. UV transilluminator PCR Purification 14. ExoSAP-IT® (USB® Products Cat No 78200 for 100 reactions) 15. 2mM MgCl2 16. Shaker Sequencing, Purification and Denaturation. 17. BigDye® Terminator Cycle Sequencing Kit v3.1 or v1.1, Applied Biosystems™ by Life Technologies™. 18. BigDye® Terminator v1.1 & v3.1 5X Sequencing Buffer, Applied Biosystems™ by Life Technologies™. 19. 125mM EDTA, pH8.0 Page 6 of 15 For Research Use Only 20. Absolute Ethanol. Each run needs freshly prepared 80% ethanol solution consisting of absolute ethanol and sterile water. DO NOT USE DENATURED ETHANOL. Other PCR and Sequencing purification procedures require validation by the user prior to use. 21. Hi-Di™ Formamide, Applied Biosystems™ by Life Technologies™, product code 4311320 22. Automated DNA Sequencer and accessories (eg Applied Biosystems™ by Life Technologies™ ABI Prism® 3730), including data collection and software. These kits have been tested and validated on the Applied Biosystems™ by Life Technologies™ 3100, 3730 and 3730xl capillary sequencers and software. The use of other sequencing platforms requires validation by the user prior to use. HLA Sequencing Analysis Software (eg Assign SBT™, version 3.5 or higher, or Assign ATF™ (Conexio Genomics Pty Ltd). 23. Sample Requirements 1. Sterile water (negative/ no template control) 2. High molecular weight human genomic DNA (concentration range of 20-100ng/µL in Tris/EDTA buffer and OD260/280> 1.8) extracted from ACD or EDTA anticoagulated whole blood specimens. Do NOT use whole blood specimens containing heparin. Warnings and Safety Precautions This kit must be used by trained and authorized laboratory personnel. All samples, equipment and reagents must be handled in accordance with good laboratory practice. In particular, all biological material should be considered as potentially infectious. The use of gloves and laboratory coats is strongly recommended. Handle and dispose of all sample material according to local and national regulatory guidelines. There are NO dangerous substances contained in any of the kit components. Do NOT use reagents beyond their expiration date. The use of kit components from different kit batches is NOT recommended. Such use may affect the assay’s performance. Use of reagents not included in this kit or not listed under “Materials, Reagents and Equipment Not Supplied” (eg alternative DNA polymerases) is NOT recommended. Such use may affect the performance of the assay. Care should be taken to prevent cross-contamination of DNA specimens. Change tips between DNA specimens wherever possible. Pre- and Post-PCR activities must be strictly physically separated. Use specifically designated equipment, reagents and laboratory coats. Ethidium bromide is a potential carcinogen. Protective gloves must always be used when preparing and handling gels. Dispose of ethidium-bromide gels and buffers according to local and national guidelines. Page 7 of 15 For Research Use Only While viewing and photographing agarose gels under UV light, always avoid direct exposure and use appropriate UV-blocking face protection, disposable gloves and laboratory coats. Procedure 1. PCR 1.1. Set up one reaction for each sample, for each loci being amplified. Include appropriate positive and negative amplification controls of known genotype and at least one no template control for each group of samples being amplified. 1.2. Prepare a fresh solution of PCR master mix each time a PCR is performed. Thaw the required number of vials of the appropriate PCR Mix. Once thawed vortex briefly. 1.3. Dispense the required amount of PCR mix and DNA polymerase into a sterile tube for the number of samples to be tested. Refer to Table 1 below. Pulse vortex the solution 3-4 times. Locus DRB3 Locus-specific PCR Mix 16.7L e.g. HLA-DRB3 MIX DNA Polymerase e.g. DNA POL-DRB3 0.3L . Table 1: Composition of the master mix required per sample. 1.4. Dispense 17L of the master mix into each reaction well. 1.5. Add 3L of sample DNA or appropriate control sample to each reaction well. Add 3L of sterile water to the no template control reaction well. 1.6. Seal the reaction wells. Mix gently by vortexing and centrifuge briefly. 1.7. Place the reaction wells into a thermal cycler and amplify the target sequence according to the thermal cycling conditions below: 95°C - 10 mins 96°C - 20 secs 60°C - 30 secs 72°C - 3 mins 33 cycles 15°C - hold 1.8. Amplification takes approximately 2.5 hours to complete. 1.9. When the PCR is completed, remove the plate from the thermal cycler and either proceed directly to gel electrophoresis or store at 4°C until required. NOTE: Purification of positive amplicons by ExoSAP-IT® treatment should occur within 24 hours of completion of PCR. Page 8 of 15 For Research Use Only 2. Agarose Gel Electrophoresis 2.1. Confirm successful amplification of the internal control amplicon in for all DNA samples tested, and the DRB3/DRB4/DRB5 target amplicon in positive control and positive DNA samples by agarose gel electrophoresis using 5L of each PCR product combined with 5L of loading buffer (alternative volumes of loading buffer should be validated prior to use). The use of 1% agarose gels is recommended. 2.2. All samples tested using the DRB3/DRB4/DRB5 PCR mixes should amplify the internal control amplicon regardless of HLA-DRB3/DRB4/DRB5 genotype. HLADRB3/DRB4/DRB5 positive samples should amplify both the internal control amplicon plus the target amplicon. The expected sizes of each amplicon are listed in Table 2. Locus Expected band sizes DRB3 target amplicon ≈ 640 bp DRB4 target amplicon ≈ 460 bp DRB5 target amplicon ≈ 470 bp Internal control band ≈ 400 bp Table 2: Expected product sizes for the DRB3/DRB4/DRB5. 3. Purification of Positive DRB3/DRB4/DRB5 PCR Product NOTE: Purification systems other than EXOSAP-IT® (eg Agencourt® AMPure® XP or column-based systems) can be used to purify these PCR products. It is strongly recommended that users validate these procedures before proceeding. If EXOSAP-IT® is to be used it is recommended that users follow the procedure described below. 3.1. Prepare a mastermix consisting of 4L of ExoSAP-IT® and 8L of 2mM MgCl2 per sample. Dispense 12L of the mastermix into the reaction well of each positive sample. Seal the tubes, vortex, and place on a shaker or gently vortex for 2 mins. Centrifuge briefly before placing into the thermal cycler. Run the thermal cycler according to the following profile: 37°C - 30mins 80°C - 15mins 4°C - hold 3.2. Upon completion, dilute the purified product 1:4 with sterile water. This dilution step will ensure that there is sufficient template to perform the sequencing reactions and ensure that the concentration of the template is sufficient to produce good quality sequence data. NOTE: A higher dilution factor (eg 1:8) may be required if consistently high signals and associated noise and artefacts are observed. Weaker PCR products may require a lower dilution factor. 3.3. ExoSAP-IT® treated samples may be stored at 4C until ready for use. Page 9 of 15 For Research Use Only 4. Sequencing Reaction NOTE: Only DRB3/DRB4/DRB5 positive samples identified by gel electrophoresis should be sequenced using the following procedure. 4.1. Table 3 lists the sequencing primers that are to be used for each locus. Locus Sequencing Primers DRB3 DRB3EX2F DRB3EX2R DRB4 DRB4EX2F DRB4EX2R DRB5 DRB5EX2F DRB5EX2R DRB5EX3F Table 3: Sequencing primers provided to sequence the positive samples for each locus. 4.2. Prepare a fresh solution of sequencing primer mix on ice each time a sequence reaction is performed. The composition and volumes for the mix are indicated per sample. Component Sequencing primer Volume 2 µL Sterile water 11.5 µL BigDye® Terminators 1 µL 5X Sequencing buffer 3.5 µL 4.3. Mix each sequencing reaction mix gently by pulse vortexing. 4.4. Dispense 18µL of the sequencing reaction mix to each appropriate reaction tube/well. 4.5. Add 2µL of purified PCR product to each appropriate well. NOTE: Care must be taken to prevent cross-contamination of sequence reactions. 4.6. Seal the reaction tubes, mix gently and centrifuge briefly to ensure that the contents are located at the base of each reaction tube. 4.7. Place the reaction tubes into a thermal cycler and run according to the following profile: Number of cycles Temperature and time 25 96°C – 10sec 50°C – 5sec 60°C – 2min 1 4°C - hold 4.8. Once the program is complete, remove the reaction tubes from the thermal cycler and either proceed directly to purification of the reaction products or store at 4C until required. It is recommended that samples are purified and run on the DNA sequencer within 24 hours. Page 10 of 15 For Research Use Only 5. Purification of Sequencing Reaction Products NOTE: Purification of the reaction products may be carried out by procedures other than the ethanol precipitation method described here. It is strongly recommended that users validate these procedures before proceeding. 5.1. Briefly centrifuge the reaction tubes/plates before proceeding. If reusable lids/caps have been used during thermal cycling label the lids/caps to avoid crosscontamination. 5.2. Carefully remove the seal. 5.3. To each reaction tube add 5µL of 125mM EDTA, pH8.0. Ensure that the EDTA reaches the base of the reaction tube. 5.4. Add 60 µL of 100% ethanol to each reaction well. Seal the plate and vortex briefly but thoroughly to ensure thorough mixing. 5.5. Pellet the extension products by centrifuging at 2000g for 45 minutes. IMMEDIATELY PROCEED TO THE NEXT STEP. If this is not possible, recentrifuge for an additional 10 minutes before proceeding. 5.6. Remove the seals to the reaction tubes and discard the supernatant by inverting the reaction tubes onto paper towel or tissues. 5.7. Place the inverted reaction tubes and paper towel or tissue into the centrifuge. Centrifuge at 350g for 1 minute to remove any residual supernatant. 5.8. Remove the reaction tubes from the centrifuge and replace in an upright position on the work bench. Discard the paper towel or tissues. 5.9. Prepare a fresh solution of 80% ethanol with absolute ethanol and sterile water. 5.10. Add 60µL of 80% ethanol to each reaction tube/well. Reseal the tubes and mix by vortexing briefly. 5.11. Spin at 2000g for 5 mins. 5.12. Repeat steps 5.6 to 5.7. 5.13. Remove the reaction tubes from the centrifuge and discard the paper towel. Reseal the reaction tubes and proceed to the denaturation step. Otherwise store at -20C for no longer than 24 hours. 6. Denaturation & Electrophoresis of Sequencing Reaction Products Denaturation of extension products 6.1. Add 12µL of Hi-Di™ Formamide to each reaction tube. Vortex and centrifuge the tubes briefly. 6.2. Incubate the reaction tubes at 98C for 5 minutes. Place the reaction tubes on ice for at least 3 minutes before being placed on the sequencer. If this is not possible, store at 4C until the reactions reach room temperature or until required. NOTE: ENSURE THAT THERE ARE NO AIR BUBBLES IN THE REACTION WELLS. THESE CAN ENTER AND DAMAGE THE CAPILLARY. Page 11 of 15 For Research Use Only 6.3. Load the reaction plate onto the automated sequencer and prepare the data collection file according to the sequencer manufacturer specifications. Electrophoresis Conditions for ABI 3730 & 3730xl automated sequencers 6.4. The following instrument parameters have been validated by the manufacturer using Big Dye® Terminator Sequencing Kit v3.1 and POP-7™. These parameters may require user validation for other polymers, sequencing chemistries and instruments. Please refer to the appropriate instrument user’s manual for detailed instructions and guidance (e.g. Dye set setting for v1.1 Big Dye® Terminator sequencing chemistry). Parameter Setting Dye set Z_BigDyeV3 Mobility file KB_3730_POP7_BDTV3 Basecaller KB.bcp Run Module Regular FastSeq50_POP7 Injection time 15 sec Collection time 3000 sec 6.5. Use the instrument’s data collection software to process the raw collected data and create the sequence files. Please refer to the appropriate instrument user’s manual for detailed instructions and guidance. 7. Editing and analysis of electropherograms The SBT Resolver™ kits were developed and validated using the Assign ATF™ software developed by Conexio Genomics Pty Ltd. Sequence analysis may also be performed using Assign SBT™ software. For more details please refer to the Conexio Genomics website (http://www.conexio-genomics.com). Limitations and Cautions It is strongly recommended that these kits are validated by the user prior to implementation in the laboratory using samples whose HLA type has been determined by other molecular based procedures. These Sequence Based Typing kits have been validated using panels of samples whose genotypes cover a broad range of alleles. However it should be noted that rare alleles, and alleles with polymorphisms in amplification and sequencing primer sites may be encountered and these may not be amplified or sequenced. A positive control (human DNA sample known to have HLA-DRB3/DRB4/DRB5), a negative control (human DNA sample known to be negative for HLADRB3/DRB4/DRB5) and no template control (sterile water) must be included on every PCR run. The positive control must produce two amplicons of the appropriate size and the resultant sequence must be in concordance with the sample’s genotype. The negative control must produce a single internal control amplicon of the appropriate size. There must be no PCR products in the no template control for each experiment. If a band is evident contamination may have occurred at some level and the run must be repeated. Occasionally there may be larger, fainter PCR products evident. These additional bands do not interfere with sequence results or quality. Page 12 of 15 For Research Use Only License The SBT Resolver™ kits contain GoTaq® Hot Start Polymerase (DNA POL) which is manufactured by Promega Corporation for distribution by Conexio Genomics Pty Ltd. Licensed to Promega under U.S. Patent Nos. 5,338,671 and 5,587,287 and their corresponding foreign patents. Troubleshooting Problem Possible cause(s) Solution No or weak PCR product Poor quality DNA Assess DNA quality by gel electrophoresis. Intact DNA should be approx 3kb with little or no evidence of smearing on gel. Re-extract DNA and repeat PCR where possible. Check concentration of DNA is between 20-100ng/L. Reextract DNA and repeat PCR where possible. Avoid the use of whole blood specimens containing heparin. Re-extract DNA and repeat PCR where possible. Repeat PCR. Ensure mastermix components are added and mixed sufficiently by vortexing. Check the thermal cycling run parameters. Check the run history to ensure that the run was not terminated prematurely. Ensure that the thermal cycler is operating according to manufacturer’s specifications and is regularly maintained. Submerge the gel in a staining bath containing 1X TBE with 0.5mg/mL ethidium bromide. Destain in 1X TBE before taking gel image. Ensure ethidium bromide is added to gel prior to pouring. Check that the appropriate kit has been used. Check the thermal cycle parameters. Check the negative control for evidence of contamination. Decontaminate work area and Insufficient quantity of DNA added to PCR. Presence of PCR inhibitors in genomic DNA DNA polymerase not added to the mastermix or insufficient mixing of mastermix prior to addition to samples. Thermal cycling problems No ethidium bromide added to the gel. Incorrect band sizes Incorrect kit used Incorrect thermal program used. PCR contamination Page 13 of 15 cycling For Research Use Only Problem Weak signal intensity of electropherograms Possible cause(s) Solution Weak PCR product Insufficient reaction products applied to sequencer Problems during purification of sequencer products Signal intensity is too high (Presence of high fluorescent peaks – artefacts) Too much PCR product Too much reaction products applied to sequencer. Noisy baseline (high background) Contaminated PCR product Amplification of closely related HLA genes Poor PCR purification Contaminated reactions sequencing Page 14 of 15 repeat PCR. Repeat PCR to identify source of contamination. Consider using a fresh kit. If the genomic DNA of a sample appears to be contaminated, re-extract or obtain an alternative source of DNA. Check gel image. Sequencing weak PCR bands is NOT recommended as the sequence quality may be insufficient for SBT. Consider using a lower dilution factor (eg 1:2, 1:3) after PCR purification. Check sequencer parameters. Injection time and voltage may need to be increased. Use extreme care when discarding the supernatant as it may dislodge the pellet. Check the gel image. Consider using a higher dilution factor following PCR purification. Check the amount of DNA polymerase used in the PCR. Check instrument parameters. Consider reducing the injection time and voltage. Refer to corrective actions listed above. Check thermal cycling parameters. Consider adjusting the parameters if an alternative thermal cycler is used. Ensure ExoSAP-IT® treatment is undertaken according to kit’s user instructions. Ensure that the PCR mixture is mixed thoroughly with ExoSAP-IT®. Ensure that all steps are taken to prevent cross contamination. Change pipette tips wherever possible. Add liquids at the top of the reaction wells. Prevent aerosols. For Research Use Only Problem Possible cause(s) Solution Contaminated sequencing primer Presence of Dye blobs Check sequence quality of the other sequencing primers and other samples using the same primer. Contaminated dye terminator Repeat sequencing with fresh mix or sequencing buffer aliquot of reagents. Poor purification of sequencing Repeat sequencing and ensure products. that purification is undertaken according to manufacturer’s instructions. Poor purification of sequencing Purify products according to products kit instructions. Ensure products are washed sufficiently with 80% ethanol. Support and Contact Details Conexio Genomics Pty Ltd 8/31 Pakenham St Fremantle 6160 Western Australia Tel: +61-422-863-227 email: [email protected] Skype: conexiocgx Website: http://www.conexio-genomics.com Page 15 of 15 Or your local distributor. For Research Use Only
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