Product no: 511.21 Batch no: TK0676 Primer Lot no: 523-x DynaMix PlusTM no: T0640 Expiry date: 2008-01 Size: 20 tests Instructions For Use Dynal AllSet+™ Minor Histocompatibility Antigen (mHA) Typing Kit For Research Use Only INSTRUCTIONS FOR USE Dynal AllSet+™ Minor Histocompatibility Antigen (mHA) Typing Kit CONTENTS Page INTENDED USE…………………………………………………………………………..….…………… 3 SUMMARY AND EXPLANATION…….........………………...………………………………………… 3 PRINCIPLES OF THE PROCEDURE ……………………………...............……………..……………… 3 REAGENTS.........................................................…................…………...….…………………………… 4 Provided in Typing kit ...………………………………..……….……………………………… 4 Storage ………………………………………………………………..……..…………..……… 4 WARNINGS AND PRECAUTIONS ………………………………………………….…………………… 5 EQUIPMENT REQUIRED BUT NOT PROVIDED BY DYNAL ……….………………………………… 5 PROCEDURE ...........……………………………….……………………………………….….…………… Sample Collection and Preparation.........................…………………………………… 6 PCR Amplification Set-up ........................…...................………...……………………… 6 PCR Amplification .................................……………………...........……………………… 6 PCR Detection using agarose gel electrophoresis …………………………………… 7 8 INTERNAL CONTROLS .................................……........…...……………………………….………… Internal Positive Control …………………………………………………………………… 9 Integrated Negative Control well ……………………………………………………… 9 9 INTERPRETATION ….……………………………………………………………………………………… 10 TROUBLESHOOTING GUIDE ...….........................……….................…...……………………………. 11 BIBLIOGRAPHY ..................………........…………...…......…..........…....…………………………… 12 WARRANTY ……………………………………………………………………………………………….. 13 TRADEMARKS USED IN THIS DOCUMENT/PRODUCT ……………..…..…………………………… 13 PATENTS USED IN THIS DOCUMENT/PRODUCT ….………………….………………………………… 13 CONTACT DETAILS ...................................................................................………………………. 14 Page 2 of 14 INSTRUCTIONS FOR USE Dynal AllSet+™ Minor Histocompatibility Antigen (mHA) Typing Kit For Research Use Only INTENDED USE DNA typing of Minor Histocompatibility antigen alleles. SUMMARY AND EXPLANATION Minor Histocompatibility (H) antigens may crucially influence the outcome of HLA-identical allogeneic stem cell transplantation 1. Typing of minor H antigens enables the identification of recipient/donor pairs eligible for adoptive immunotherapy and of pairs with increased risk for GvHD. The minor H antigen genotyping SSP Kit has been designed to address these issues in the minor H antigen component of the 14th International Histocompatibility and Immunogenetics Workshop (www.lumc.nl/ihiw14). The kit is a PCR-based method designed to detect the alleles of 11 different minor H antigens 2. The minor H antigen typing protocol has been constructed to match the common HLA typing protocols. It can therefore easily be incorporated in routine tissue typing procedures. The method has been tested on DNA from EBV-LCL that were typed using the relevant minor H antigen specific T cell clones. PRINCIPLES OF THE PROCEDURE Almost all DNA-based typing technologies utilize the PCR technique 3 to amplify the target to be investigated. In most DNA-based methods of tissue typing, the PCR technique is used as a pre-typing amplification step to increase the amount of target DNA. The typing process then requires a post- amplification step to discriminate between the different alleles. Unlike other PCR based methods, the SSP Methodology 4,5,6,7 employed by Dynal Biotech Ltd – AllSet+™ SSP, discriminates between the different alleles during the PCR process. This shortens the post-amplification processing time to a simple gel electrophoresis detection step. The Dynal SSP method is a PCR based technique, which uses Sequence Specific Primers (SSP), for DNA based typing. The Dynal SSP products consist of panels of primer mixes where each primer mix contains one or more specific primer pairs, i.e. the allele- and/or group-specific primers, as well as a control primer pair matching non-allelic sequences in the samples. The control primer pair functions as an internal PCR control to verify the efficiency of the PCR amplifications. Dynal SSP is based on the principle that a perfectly matched primer will be more efficiently used in the PCR reaction than a primer with one or several mismatches at its 3’ –end. The specificity of the typing system is part of the PCR amplification step, and post-amplification processing of the samples is reduced to a minimum. The assignment of alleles merely consists of determining whether amplification has occurred or not, i.e. visualisation and detection of the amplification by agarose gel electrophoresis. The PCR-SSP technique provides a high degree of resolution because each primer pair identifies two linked, cis-located polymorphic sites. The method provides high sensitivity, specificity and reproducibility. Page 3 of 14 INSTRUCTIONS FOR USE Dynal AllSet+™ Minor Histocompatibility Antigen (mHA) Typing Kit REAGENTS Reagents provided in the Dynal AllSet+™ Minor Histocompatibility Antigen (mHA) Typing Kit Item Description Each well in the PCR tray contains a dried SSP primer 1. PCR Trays with pre-aliquoted and solution consisting of allele- and/or group-specific dried SSP Primer Mixes primers as well as a control primer pair matching non- allelic sequences. The control primer pair amplifies a fragment of a conserved gene present in all samples. There is an integrated negative control in well # 21. 2. PCR Caps Caps for sealing the PCR Trays. 3. Master Mix 3 vials each containing 1.8ml Ready to use 1 x concentration. The Master Mix has been optimised for use with AmpliTaq® DNA Polymerase (Roche Molecular Systems, Inc.) supplied at 5i.u./μl Formulation information available from [email protected] Combined Instructions For Use and Batch Specific 4. Combined Product Instructions Product Information (BSPI) booklet. For Use and Batch Specific Product Information booklet 5. Interpretation worksheet A worksheet to determine which allele or group of alleles the respective primer mixes identify. Storage 1. PCR Trays and Master Mix must be stored at 2-8˚C. 2. Do not freeze reagents. 3. If correctly stored, these reagents and primer mixes are stable until the expiration date indicated on this Batch Specific Instructions For Use Booklet. Page 4 of 14 INSTRUCTIONS FOR USE Dynal AllSet+™ Minor Histocompatibility Antigen (mHA) Typing Kit WARNINGS AND PRECAUTIONS 1. For Research Use Only. 2. All work with Dynal AllSet+™ Products should be performed using Good Laboratory Practice and according to local guidelines e.g. EFI standards, CPA guidelines. 3. Biohazard warning Handle all samples as if capable of transmitting disease. All work should be performed wearing appropriate gloves and protection. 4. Biohazard warning Screw cap tubes should be used for specimen preparation to prevent specimen splashing and potential contamination. 5. Biohazard warning: All specimens should be handled as if infectious using safe laboratory procedures such as those outlined in Biosafety in Microbiological and Biomedical Laboratories 8 and in the NCCLS Document M29-T 9. Thoroughly clean and disinfect all work surfaces with 1% bleach (NaClO). Autoclave any equipment or materials that have come in contact with clinical specimens before discarding. 6. Biohazard warning: The ethidium bromide used for staining of DNA is a potential carcinogen. Always wear nitrile gloves when handling stained gels and ethidium bromide. 7. Caution: Wear UV-Blocking eye protection and do not view UV light source directly with naked eye when viewing or photographing gels. 8. Caution: Pipettes used for post-PCR manipulations should not be used for pre-PCR manipulations. 9. Caution: Contamination. Avoid microbial contamination of reagents when removing aliquots from reagent bottles. Always wear gloves to avoid any risk of sample contamination. The use of sterile disposable pipettes and filter pipette tips is recommended. Do Not use reagents with evidence of turbidity or microbial contamination. 10. Caution Disposal: Dispose of unused reagents and waste in accordance with country, federal, state and local regulations. 11. Material Safety Data Sheet (MSDS) is available to download at www.dynalbiotech.com or on request from your local Dynal Biotech Office. (See contact details listed on last page of this insert.) EQUIPMENT REQUIRED BUT NOT PROVIDED BY DYNAL BIOTECH: 1. Manual pipetting devices (e.g Eppendorf, Gilson) 2. Disposable filter pipette tips for above 3. Vortex mixer 4. Microcentrifuge 5. PCR tray microtube storage rack 6. Perkin-Elmer GeneAmp® PCR system 9600 or 9700 or any licensed thermalcycler of equivalent specification 7. Hot-plate / microwave for heating agarose solutions 8. Electrophoresis apparatus (e.g ABgene Electro-Fast® Stretch 108 Complete, AB-0708) 9. UV transilluminator 10. Gel Photographic/Image documentation system 11. Recombinant Taq Polymerase (Dynal recommends AmpliTaq®) 12. Electrophoresis grade agarose 13. Ethidium bromide Page 5 of 14 INSTRUCTIONS FOR USE Dynal AllSet+™ Minor Histocompatibility Antigen (mHA) Typing Kit PROCEDURE NOTE: This procedure should be performed in two areas of the laboratory (Pre-PCR Amplification and Post-PCR Amplification) as indicated in the following instructions. SAMPLE COLLECTION AND PREPARATION Caution: Handle all specimens as if they are capable of transmitting infectious agents. a) THE USE OF HEPARINISED BLOOD IS NOT RECOMMENDED 10. b) DNA can be extracted from human nucleated cells by any preferred method. c)