Instructions for Use

Transcription

1 Instructions for Use SSP/SNP typing within the central MHC (Gamma block) PCR-SSP Tray Format (6 and 25 tests) Version No: 3.0 Issue Date: July 2016 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 20 Collie St. Fremantle 6160 Western Australia Australia Page 1 of 16 For Research Use Only

2 Contents PRINCIPLE... 3 KIT CONTENTS... 4 STORAGE REQUIREMENTS... 5 MATERIALS, REAGENTS AND EQUIPMENT NOT SUPPLIED... 5 SAMPLE REQUIREMENTS... 5 CONTROLS... 5 Internal Controls... 5 No Primer Control (NPC)... 5 No Template Control (NTC)... 6 Positive Control... 6 Worksheets... 6 Gamma-Type Matching Worksheet... 6 GAMMA-TYPE WORKFLOW... 8 PROCEDURE PCR AGAROSE GEL ELECTROPHORESIS AND INTERPRETATION LIMITATIONS AND CAUTIONS TROUBLESHOOTING SUPPORT AND CONTACT DETAILS APPENDIX A GAMMA-TYPE BANDING PATTERN APPENDIX B - GAMMA-TYPE GEL IMAGES APPENDIX C - GAMMA-TYPE MATCHING WORKSHEET Page 2 of 16 For Research Use Only

3 Principle It has been shown that Major Histocompatibility Complex (MHC) haplotype matching of patients and donors reduces the risk of acute Graft-versus-host Disease (agvhd) in hematopoietic stem cell transplants (HSCT). The MHC is usually inherited as a single locus, however family studies have shown that recombination occurs at specific sites across the MHC resulting in a 4 x genomic block structure. The four major MHC blocks are each several hundred kilo bases in length and contain many genes. HLA-A is located in the Alpha block, HLA-B and HLA-C in the Beta block, the Bf, C2 and C4 genes are in the Gamma block and the HLA-DRB and HLA-DQB genes are in the Delta block. The HLA genes are the most polymorphic genes within their blocks and serve as block "markers". Therefore Human Leukocyte Antigen (HLA) typing is the current method used to infer MHC ancestral haplotypes (AH) when matching unrelated patients and donors. HLA alleles however, are not always haplotype specific, so haplotype matching cannot always be predicted. The Gamma block, as well as the central MHC region between the Gamma and Beta blocks, contain many inflammatory and immune regulatory genes. However the Gamma block is rarely typed, leaving a large region of the central MHC uncharacterised during transplantation. The Gamma block is a key locus when predicting haplotype matching. Gamma-Type + HLA-B + HLA-C matching indicates complete sequence matching across the Beta block-central MHCgamma block region. Gamma-Type mismatching indicates the presence of recombinant haplotypes and potential genetic mismatching across the Beta Gamma block region. Given that the central MHC contains pro-inflammatory cytokine and immune response genes and that it has been implicated in many MHC disease associations, it can be speculated that this region may also be key to outcome in unrelated HSCT. The Gamma-Type kits consists of a panel of sequence specific primers (SSP) for SNPs located in the Gamma block of the MHC that will assist in haplotype matching between patients and unrelated stem cell donors. Gamma-Type is a research use only tool and is intended to be used to provide additional haplotype matching information between HLA typed stem cell donors and patients. In addition to haplotype matching for unrelated stem cell transplantation, the haplotype specific SNPs can also be used in MHC disease gene mapping studies and to assist in the identification of non-hla transplantation genes. Page 3 of 16 For Research Use Only

4 Kit Contents Kit Name Catalogue Number KD-PD8.0-1(6) Gamma-Type KD-PD8.0-1(25) 6 tests 25 tests Kit Contents 6x 1 test PCR-SSP Trays Each tray contains: - 25 wells of PCR-SSP Mix* (8µL in each well) - 2 wells of No Amplification Control Mix (8µL in well 26 and 27) For the tray format please see below. DNA POL Gamma-Type 1 vial sufficient for 150 reactions. NTC POL Gamma-Type 1 vial sufficient for 6 reactions. 25x 1 test PCR-SSP Trays Each tray contains: - 25 wells of PCR-SSP Mix* (8µL in each well) - 2 wells of No Amplification Control Mix (8µL in well 26 and 27) For the tray format please see below. DNA POL Gamma-Type 1 vial sufficient for 650 reactions. NTC POL Gamma-Type 1 vial sufficient for 25 reactions. PCR-SSP Trays The PCR-SSP mixes are aliquotted into the PCR-SSP trays in the following format: A Mix 01 Mix 09 Mix 17 Mix 25 B Mix 02 Mix 10 Mix 18 NPC, 26 C Mix 03 Mix 11 Mix 19 NTC, 27 D Mix 04 Mix 12 Mix 20 Empty E Mix 05 Mix 13 Mix 21 Empty F Mix 06 Mix 14 Mix 22 Empty G Mix 07 Mix 15 Mix 23 Empty H Mix 08 Mix 16 Mix 24 Empty Sample 1 * Contains PCR buffer, dntps, MgCl2, sequence specific primers (SSPs) and green loading buffer. Page 4 of 16 For Research Use Only

5 Storage Requirements Gamma-Type kits must be stored at -20C immediately upon receipt in a temperature stable freezer. When stored at -20C, the kit components can be used until the expiry date displayed on the outer kit label. Materials, Reagents and Equipment Not Supplied 1. Electronic or mechanical pipettes and aerosol-resistant tips 2. Thermal cycler with heated lid These kits have been optimised and validated using the following thermal cyclers: MJ Research PTC 225 DNA Engine DYAD Eppendorf Mastercycler Pro. Applied Biosystems Veriti NOTE: Use of other thermal cyclers with these kits will require validation by the user. 3. Sterile work area such as biological safety cabinet or hood. 4. Sterile 1.5mL tubes 5. Table top centrifuge with plate adapters 6. Vortex 7. Agarose gel electrophoresis apparatus 8. 2% agarose (molecular biology grade) TBE gel containing 0.1µg/mL ethidium bromide 9. PCR Marker suitable to cover range of bp 10. UV transilluminator Sample Requirements High molecular weight human genomic DNA (concentration range of ng/µL in Tris/EDTA buffer and OD 260/280> 1.8) extracted from ACD or EDTA anticoagulated whole blood specimens. Do NOT use whole blood specimens containing heparin. Controls Internal Controls Each Gamma-Type PCR-SSP mix contains SSP s for a SNP target as well as a pair of internal control amplification primers. Amplification of the internal control must always be detected in negative reactions (those that do not amplify for the target) to ensure reaction conditions are optimal. The internal control band may drop out in presence of the target amplicon (positive reactions). No Primer Control (NPC) Mix 26 of the Gamma-Type SSP trays is a negative (or no amplification ) control and should be set up for each sample tested in the same manner as all other mixes. No amplification of template DNA is expected in the negative control since amplification primers are not present. The negative control is important to ensure there is no cross-contamination being observed Page 5 of 16 For Research Use Only

6 between mixes (i.e. there is no primer carry over during dispensing of DNA) resulting in false positives. No Template Control (NTC) Mix 27 of the Gamma-Type SSP trays is a negative (or no template ) control and should be set up for each sample tested. The NTC reaction is to be tested using the water (or the reagent used to dilute/elute DNA during DNA extraction/preparation) to detect contamination. This mix contains the internal control primers used in mixes 1 to 25 of the Gamma-Type SSP tray. Positive Control It is recommended that a positive control with a known Gamma-Type profile be included at least once with each new batch of kits to ensure accurate SNP calling is achieved. Please refer to the Conexio Genomics website ( for Gamma-Type TM profiles of UCLA QAP samples that can be used as positive controls. Worksheets Gamma-Type worksheets are available to download from the Conexio Genomics website ( Gamma-Type Matching Worksheet The Gamma-Type Matching worksheet allows users to easily compare a single patient with up to 12 donors for all SNPs. There is space on the worksheet to record the sample information (for both patient and donors), as well as the kit and run details. Users can insert their patient and donor gel images into the worksheet and enter results using the scoring table provided. When all the results are entered, the matching status of the various donors to the patient can be determined using the <Compare Results> button. 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 (e.g. 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. Page 6 of 16 For Research Use Only

7 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. 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. Page 7 of 16 For Research Use Only

8 Gamma-Type Workflow Page 8 of 16 For Research Use Only

9 Procedure 1. PCR 1.1. Each PCR-SSP tray contains enough Gamma-Type mix for a single sample. Quickly thaw the Gamma-Type mixes / reaction tray at room temperature. Once thawed, vortex briefly and pulse spin the tray to ensure all mixes are brought down to the bottom of the wells Prepare a mixture of genomic DNA and DNA Polymerase (DNA POL Gamma- Type ) for each sample to be typed according to the table below. This should be prepared fresh for every new PCR. Pulse vortex the solution 3-4 times to mix. Table 1: Composition of the DNA/polymerase mixture required per sample. Reagent Genomic DNA DNA POL Gamma-Type Volume 57.5L 2.5L 1.3. Dispense 2L of the DNA/polymerase mixture into reaction wells 1 to 26 (refer to the table shown in Kit Contents for the layout of the reaction wells and their contents) To the NTC ( no template control ) reaction (i.e. well 27) dispense 1µL of NTC POL - Gamma-Type and 1µL of water Seal the reaction wells. Mix gently and centrifuge briefly Place the reaction tray into a thermal cycler and amplify according to the thermal cycling conditions below: 95 C - 10 mins 96 C - 20 secs 61 C - 30 secs 28 cycles 72 C - 3 mins 96 C - 20 secs 56 C - 30 secs 5 cycles 72 C - 3 mins 15 C - hold 1.7. Amplification takes approximately 2.5 hours to complete. When the PCR is complete, remove the plate from the thermal cycler and either proceed directly to gel electrophoresis or store at 4 C until required. Page 9 of 16 For Research Use Only

10 2. Agarose Gel Electrophoresis and Interpretation 2.1. Confirm amplification of the internal control and the target amplicons by agarose gel electrophoresis using 2L of each PCR product. The addition of loading buffer is not required as this is already contained within the PCR-SSP mixes. The use of 2% agarose gel is recommended and PCR products should be electrophoresed for 30 minutes at 150 volts to ensure sufficient band separation is achieved There must be no PCR products in the no amplification control wells (i.e. wells 26 and 27) for each sample tested. If a band is evident in either well, contamination may have occurred at some level and the run must be repeated All negative Gamma-Type reactions should amplify the internal control amplicon. If neither the target, nor internal control amplicon are evident, the reaction cannot be interpreted and a failure (0) should be recorded on the relevant Gamma-Type worksheet A positive Gamma-Type reaction is observed and recorded when the target amplicon is amplified. Check the expected size of each target amplicon (refer to Table 2) when scoring the reactions. NOTE: Positive reactions may have a strong target amplicon present with a very weak or absent internal control. This is an acceptable positive reaction (See Appendix A). When scoring reactions, record results using the number 1 for negative reactions and 2 for positive reactions. Any failed reactions (as described in point 2.3) should be recorded as Patient Gamma-Type profiles may be compared with donor Gamma-Type profiles to assist in the prediction of Gamma block matching. In order to do this, pertinent sample information including the Name, Sample ID and DNA concentration, should be recorded on the Gamma-Type Matching Worksheet for each patient and potential donor tested (See Appendix C). Copies of the Gamma-Type gel images may be imported into the worksheet. The gel image can then be interpreted by scoring positive reactions where the target band is present with 2 and negative reactions where only the internal control band is present with 1. Failed or no amplification reactions, where both the target and internal control are absent, are scored with a 0. These results should be entered in the Test Results row of the Matching Worksheet for the patient and donors Once all results have been entered and scored, click the <Compare Results> button within the Gamma-Type Result Summary table. The worksheet will then automatically determine if any of the donors typed/entered are Gamma-Type matched to the patient. Please note that in some circumstances you may receive an Indeterminate result or an error message. This may be due to an incomplete result for either the patient or donors, or if an unacceptable result is recorded. The worksheet cannot analyse incomplete or unacceptable patient results. As such tests must be repeated if contamination is detected or any reactions fail. For further information please refer to the Instructions tab of the Gamma-Type Matching Worksheet or the Troubleshooting section of this IFU. Page 10 of 16 For Research Use Only

11 Table 2: Expected product sizes for the Gamma-Type mixes. Gamma-Type Mix SSP Target Amplicon Size Internal Control Amplicon Size bp 450 bp bp 450 bp bp 450 bp bp 450 bp bp 450 bp bp 450 bp bp 450 bp bp 300 bp bp 450 bp bp 450 bp bp 450 bp bp 450 bp bp 450 bp bp 450 bp bp 300 bp bp 450 bp bp 300 bp bp 300 bp bp 300 bp bp 300 bp bp 300 bp bp 300 bp bp 450 bp bp 300 bp bp 300 bp No Amp. No Amp. No Amp. No Amp. Page 11 of 16 For Research Use Only

12 Limitations and Cautions It is strongly recommended that these kits are validated by the user prior to implementation in the laboratory using samples that have been HLA typed by other molecular based procedures and for which ancestral haplotypes can be predicted. Please refer to the Conexio Genomics website ( for Gamma-Type TM profiles of UCLA QAP samples that can be used as positive controls. All of the Gamma-Type mixes should be set up for each sample to be tested. Mixes 8 and 15 are used as amplification controls. All samples should be positive for at least one of the two amplification control assays. In some instances the Gamma-Type Mixes 7 and 17 may produce a larger, fainter band/s. This should not interfere with identification of a true positive target band. If this occurs please refer to Table 2 to identify the target amplicon size. In some instances weak false positive amplification of the target band may occur for some of the Gamma-Type Mixes. In such cases the false positive bands will appear much weaker than true positive target bands (see positive control mixes 8 and/or 15 as an indicator) or the internal control bands. It is recommended that the band intensities are compared between the patient and donor to ensure Gamma-Type profile matching. If unsure, the reaction should be recorded as indeterminant (score as 0 on the matching worksheet). Troubleshooting Problem Possible cause(s) Solution Some separation is visible within the mixes upon thawing No or weak PCR product Separation of PCR mix components may have occurred during transportation Poor quality DNA Insufficient quantity of DNA added to PCR. Presence of PCR inhibitors in genomic DNA DNA polymerase not added to the master mix or insufficient mixing of master mix prior to addition to samples. Thaw the PCR-SSP trays and vortex briefly but thoroughly to mix and then briefly spin the plate before use. Separation of components does not affect the performance of this kit. Assess DNA quality by gel electrophoresis. Intact genomic 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 ng/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 master mix components are added and mixed sufficiently by vortexing. Page 12 of 16 For Research Use Only

13 Problem Possible cause(s) Solution Thermal cycling problems No ethidium bromide added to the gel. 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. De-stain in 1X TBE before taking gel image. Ensure ethidium bromide is added to gel prior to pouring. Incorrect band sizes Incorrect plate orientation. Check that the correct plate orientation was used during PCR setup and/or gel electrophoresis. Incorrect thermal cycling program used. PCR contamination Check the thermal cycle parameters. 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. Support and Contact Details Conexio Genomics Pty Ltd PO Box 1294 Fremantle 6959 Western Australia Tel: support@conexio-genomics.com Skype: conexiocgx Website: Or your local distributor. Conexio is a trademark of Conexio 4 Pty Ltd. Page 13 of 16 For Research Use Only

14 Appendix A Gamma-Type Banding Pattern Note: Wells 26 and 27 are not shown. These are negative controls; no bands should be observed. Page 14 of 16 For Research Use Only

15 Appendix B - Gamma-Type Gel Images Positive and negative profile of PCR-SSP Mix Note: Wells 26 and 27 are not shown. These are negative controls; no bands should be observed. Page 15 of 16 For Research Use Only

16 Appendix C - Gamma-Type Matching Worksheet The Gamma-Type Matching Worksheet is available to download from the Conexio Genomics website ( The worksheet allows users to automatically compare patient results with the results of multiple donor. See section Agarose Gel Electrophoresis and Interpretation for more details. Page 16 of 16 For Research Use Only