PRODUCTINSERT SSP Minor Histocompatibility Antigen Primer Sets
Contents
1. 4A One Lambda Inc 21001 Kittridge Street Canoga Park CA 91303 2801 Tel 818 702 0042 Fax 818 702 6904 WEB www onelambda com PRODUCT INSERT SSP Minor Histocompatibility Antigen Primer Sets Catalog BSSPMHAG For Research Use Only Not for use in diagnostic procedures STORE REAGENTS AT TEMPERATURE INDICATED ON PACKAGE USE BEFORE EXPIRATION DATE Warning Use only the D mix packaged with this Liquid Bulk Primer Set DIRECTIONS FOR USE Formulation Use the following formulation to prepare the reagents for your test 1 Use 2 ul of primer set per PCR reaction Use 1 ul of the test sample and 7 ul D mix per reaction 2 3 Use 05 ul at 5 units ul of recombinant Taq polymerase per reaction 4 Perform standard PCR with the Micro SSP PCR program OLI 1 as specified below INTENDED USE DNA typing of the Minor Histocompatibility locus HA 1 SUMMARY AND EXPLANATION Successful marrow stem cell engraftment requires that bone marrow BM donor recipient pairs be matched for HLA A B DR and DQ Nevertheless among HLA genotype identical donor recipient pairs acute graft versus host disease GvHD occurs in 15 35 of cases after allogeneic BM transplantation depending on the age of the recipient and the amount of T cell depletion of the graft The occurrence of acute GVHD has been ascribed to mismatching the minor histocompatibility antigens mHags encoded by the HA 1 locus The HA 1 locus is co2. amplification in every test reaction The amount of each primer is adjusted for optimal amplification of 100 ng of sample DNA when used in conjunction with the Primer Set D mix the prescribed amount of recombinant Taq polymerase and the PCR reaction profile detailed below B Warning or Caution 1 For research use only Not for use in diagnostic procedures 2 Limit refreezing of primers no more than twice 3 Pipettes used for Post PCR manipulations should not be used for Pre PCR manipulations 4 Biohazard Warning The ethidium bromide used for staining of DNA is a potential carcinogen Always wear gloves when handling stained gels 5 Biohazard Warning All blood products should be treated as potentially infectious 6 Caution Wear UV blocking eye protection and do not view UV light source directly when viewing or photographing gels 7 Aliquot D mix into convenient to use volumes Do not refreeze more than twice C Storage Instructions Store reagents at temperature indicated on package Use before printed expiration date D Purification for Treatment Required for Use See Directions for Use below E Instability Indications 1 Any primer set with a vial that is damaged should be considered unusable 2 Ifsalts have precipitated out of the solution in the D mix aliquots during shipping or storage re dissolve by extended vortexing at room temperature 20 25 C 3 D mix aliquots upon thawing at room tem
3. may be adversely affected by various factors pipetting errors poor DNA quality presence of inhibitors etc an internal control primer pair is included in every PCR reaction The control primer pair amplifies a conserved region of the human B globin gene which is present in all DNA samples and is used to verify the integrity of the PCR reaction In the presence of a positive typing band specific amplification of an HA 1 allele the product of the internal control primer pair may be weak or absent due to the differences in concentration and melting temperatures between the specific primer pairs and the internal control primer pair The amplified DNA fragments of the specific HLA primer pairs are smaller than the product of the internal control primer pair but larger than the diffuse unincorporated primer band Thus a positive reaction for a specific HA 1 allele is visualized on the gel as an amplified DNA fragment between the internal control product band and the unincorporated primer band see Results Section Page 1 of 4 REAGENTS A Identification The SSP Minor Histocompatibility Antigen Primer Sets provide sequence specific oligonucleotide primers for amplification of alleles and the human B globin gene by the polymerase chain reaction PCR and specially formulated dNTP buffer mix Primer Set D mix The internal control PCR product amplified from the human B globin gene is the most likely contaminating PCR product due to its
4. Histidine 345ACACT349 HA 1R Arginine 345ACACT349 3 Recognition Site 500CTGCA504 Approx Size Base Pairs 500TTGCG504 Based on the KIAA0223 cDNA Sequence Information Gene Bank Accession Number D86976 REFERENCES 1 Goulmy E Schipper R Pool J et al Mismatches of minor histocompatibility antigens between HLA identical donors and recipients and the development of graft versus host disease after bone marrow transplantation New Engl Med 1996 334 281 5 2 Goulmy E Human minor histocompatibility antigens Curr Opin Immunol 1996 8 75 81 3 Den Haan JMM Meadows LM Wang W et al The minor histocompatibility antigen HA 1 a diallelic gene with a single amino acid polymorphism Science 1998 279 1054 7 TRADEMARKS USED IN THIS DOCUMENT PRODUCT ARMS Zeneca Limited FMC SeaKem FMC Corporation Fotodyne FOTO UVX 21 FOTODYNE Incorporated Gene Amp Hoffmann LaRoche Inc Gilson and Pipetman Rainin Instrument Co Inc Rev 2 BSSPMHAG PI DOC PATENTS USED IN THIS DOCUMENT Nothing in this publication should be construed as an authorization or an implicit license to practice PCR under any patents held by Hoffmann LaRoche Inc Gene Amp PCR Process is covered by U S Patent Nos 4 683 202 4 683 195 4 800 159 and 4 965 188 owned by F Hoffmann La Roche Inc The use of PCR for in vitro diagnostic procedures requires a license that can be obtained by contacting PCR Licensing Manager Director of Li
5. amples should not be re suspended in solutions containing chelating agents such as EDTA above 0 5 mM in concentration 4 DNA samples may be used immediately after isolation or stored at 20 C or below for extended periods of time over 1 year with no adverse effects on results 5 DNA samples should be shipped at 4 C or below to preserve their integrity during transport PROCEDURE A Materials Provided 1 2 vials 60 ul primer 2ul per test each 2 1000 ul Primer Set D mix 7 ul per test Note The volumes provided are slightly more than the amount required for testing This is to account for inadvertent losses which may result from pipetting B Materials Required but Not Provided e Pipetting devices such as Gilson P20 Gilson P200 Pipetman Disposable pipette tips Vortex mixer Microcentrifuge PCR tray microtube storage rack and cover Robbins Scientific Cat 1044 39 5 e Thermocycler for PCR with 96 well format and tray retainer for 0 2 ml thin walled reaction tubes e Hot plate or microwave oven for heating agarose solution e Electrophoresis apparatus power supply 150V minimum capacity Rev 2 BSSPMHAG PI DOC e UV transilluminator example Fotodyne FOTO UV 21 e Photographic or image documentation system e 1x TBE buffer 89mM Tris Borate 2 mM disodium EDTA pH 8 0 with 0 5 ug ml ethidium bromide or SXTBE Buffer with Ethidium bromide OLI Cat S5XTBE100 e Electrophoresis grade agarose e
6. censing F Hoffmann LaRoche Roche Molecular Systems Building 222 350 1145 Atlantic Avenue CH 4500 Basel Alameda CA 94501 Switzerland USA SSP technology is licensed from Zeneca Limited through its Zeneca Diagnostics business Blacklands Way Abingdon Business Park Abingdon Oxfordshire England OX14 IDY and covered under the following patents held by Zeneca Corporation European Patent No 0 332 435 B1 United States Patent No 5 595 890 entitled Method of detecting nucleotide sequences and Canadian Patent No 1323592 and corresponding patents and patent applications worldwide The SSP Minor Histocompatibility Antigen Primer Sets are manufactured and distributed by One Lambda Inc 21001 Kittridge Street Canoga Park CA 91303 USA Page 4 of 4
7. g FMC Seakem LE C Step by Step Procedures Test Procedure Refer to Directions for Use LIMITATIONS OF THE PROCEDURE PCR SSP is a dynamic process requiring highly controlled conditions to insure discriminatory amplification The procedure provided this product must be strictly followed The extracted sample DNA provides the template for the specific amplification process and thus must have its concentration and purity within the ranges specified in the procedure All instruments e g PCR machine pipetting devices must be calibrated according to the manufacturer s recommendations RESULTS A Data Analysis 1 An internal control band slower migrating should always be visible in negative wells except in the negative control well as a control for successful amplification Non amplification of any well can void test results 2 A faster migrating positive typing band will be observed on the electrophoretic gel if a specific mHag gene was amplified during PCR This indicates a positive test result 3 The internal control band may be weak or absent in positive wells 4 A positive band indicates the presence of the allele identified in the table on the next page Primer Recognition Sites B Gel Interpretation Positive Negative Non Reaction Reaction amplification Well Internal Control Band e ns Positive Typing a Primer Band Page 3 of 4 PRIMER RECOGNITION SITES 5 Recognition Site HA 1H
8. mposed of two alleles HA 1 and HA 1 which differ in two nucleotides resulting in a single amino acid substitution Disparities between mHags of otherwise HLA identical bone marrow donor and recipient pairs can elicit strong major histocompatibility complex restricted cytotoxic and proliferative T cell responses which can be measured in vitro It has been demonstrated that HLA A 0201 molecules have a high affinity for the HA 1 peptide and this mHag peptide MHC complex is recognized by HA 1 specific cytotoxic T cells 2 PRINCIPLE The PCR SSP methodology is based on the principle that completely matched oligonucleotide primers are more efficiently used in amplifying a target sequence than a mismatched oligonucleotide primer by recombinant Taq polymerase Primer pairs are designed to have perfect matches only with a single allele or group of alleles Under strictly controlled PCR conditions perfectly matched primer pairs Rev 2 BSSPMHAG PI DOC result in the amplification of target sequences i e a positive result while mismatched primer pairs do not result in amplification i e a negative result After the PCR process the amplified DNA fragments are separated by agarose gel electrophoresis and visualized by staining with ethidium bromide and exposure to ultraviolet light Interpretation of PCR SSP results is based on the presence or absence of specific amplified DNA fragment Since the amplification during the PCR reaction
9. perature 20 25 C should be pink to light purple in color Any D mix aliquot without the specified coloration should be considered unusable Rev 2 BSSPMHAG PI DOC INSTRUMENT REQUIREMENTS 1 Programming the Thermocycler The following program is designed for use only on the Perkin Elmer 9600 or 9700 thermocyclers If you have a different make and or model thermocycler you may need to adapt the programming to your machine Program your thermocycler before starting the Directions for Use Micro SSP PCR Program OLI 1 of Step Temp Time Cycles C sec 1 1 96 130 2 63 60 9 1 96 10 2 63 60 20 1 96 10 2 59 50 3 72 30 End 1 4 For specific thermocycler information refer to the manufacturer s user manual 2 2 5 Agarose Gel Preparation for Micro SSP Gel System OLI Cat MGS108 a To set up e Slide the locking pin on the base to the open position e Insert the gel box into the base by sliding the locking pin into the locked position e Use the leveling bubble and the three height adjustable legs to level the base b Orient and fully insert the 14 gel combs into the gel comb holder c To 100 ml of 1x Tris Borate EDTA buffer 1xTBE with 0 5 ug ml ethidium bromide in a 500 ml glass bottle add 2 5 g electrophoresis grade agarose Heat until a homogeneous solution is formed d Add 30 ml of the gel solution to the gel box Make sure the agarose covers the entire su
10. rface evenly by tilting the gel box back and forth immediately after the gel solution is added Quickly place the gel comb holder on the filled gel box by matching the color coding Allow to set for 15 minutes e Remove the gel combs by lifting the gel comb holder while holding the base Add 10 ml of 1X TBE containing 0 5 ug ml ethidium bromide evenly across the gel to fill every well Page 2 of 4 3 Gel Electrophoresis for Micro SSP Gel System OLI Cat MGS108 a Transfer each PCR reaction 10 ul in sequence to the 2 5 agarose gel Make sure to transfer all samples in the proper sequence No addition of electrophoresis dye is necessary Use of an 8 or 12 channel Pipetman is recommended b Cover the gel box with the gel box cover by matching the color coded sides Electrophorese the samples at 140 150 volts until the red tracking dye has migrated about 0 5 cm into the gel approximately 3 5 minutes depending on the agarose used Remove the cover c Slide the locking pin on the base to the open position and remove the gel box Transfer the gel box to a UV transilluminator Photograph the completed gel SPECIMEN COLLECTION AND PREPARATION 1 DNA can be purified from human leukocytes by any preferred method 2 The DNA sample to be used for PCR SSP analysis should be re suspended in sterile water or in 10 mM Tris HCl pH 8 0 9 0 at a concentration of 25 200 ng ul with the A260 A280 ratio of 1 65 1 80 3 S
Download Pdf Manuals
Related Search