Micro SSP HLA DNA Typing Trays, ENGLISH VERSION
Contents
1. 0 5 g ml ethidium bromide or 5XTBE Buffer with ethidium bromide OLI Cat 5XTBE100 Electrophoresis grade agarose e g FMC Seakem LE C Step by step procedure See Directions For Use below DIRECTIONS FOR USE A Sample Preparation 1 Purify genomic DNA from leukocyte sample by method of choice Final DNA concentration should be 25 200ng l 100ng l is optimal with the A260 A280 ratio between 1 65 1 80 2 For specific information on sample preparation and storage see Specimen Collection and Preparation above 3 Perform PCR on the purified DNA sample using a Micro SSP HLA DNA Typing Tray or store DNA sample at 20 C or below until ready to type B Reagent Equipment Preparation 1 Program a thermal cycler to run the One Lambda PCR program See Instrument Requirements above 2 Have available recombinant Taq polymerase 5 units l Store at 20 C 3 Prepare electrophoresis gel 2 5 agarose using the Micro SSP Gel System Cat MGS108 or with at least 96 sample wells space rows of wells at least 1 cm apart C Instructions for Pipetting Taq Polymerase One Lambda s quality control tests indicate that the volume shown on the Taq polymerase label is more than sufficient for the number of tests indicated in this product If additional Taq is required it must be ordered directly from F Hoffmann La Roche or their representative To avoid waste please follow the simple instructions lis2. 18 Photograph the gel on a UV transilluminator 19 Interpret the typing results using the worksheet provided with the trays or with the assistance of the HLA software available from One Lambda Inc Note When One Lambda s DNA typing trays fill only a few rows of the electrophoresis gel it is possible to place several samples on one gel Care should be used to record sample positions on the gel RESULTS Allele Groups DR 72 Serology Micro SSP DRB1 01 12 12 DRB1 15 24 24 DRB1 16 2 2 DRB1 0301 0304 6 6 DRB1 0302 0303 6 6 DRB1 04 14 14 DRB1 11 23a 24 DRB1 12 5 5 DRB1 13 17 17 DRB1 14 11b 10 DRB1 07 16 16 DRB1 08 7 7 DRB1 09 5 5 DRB1 10 5 5 DRB5 26 26 DRB3 57c 56 DRB4 32d 33 DQB1 05 34 34 DQB1 06 32 32 DQB1 02 24 24 DQB1 0301 0304 22 22 a b The discrepancies on DRB1 11 and DRB1 14 are based on one sample in which serology assigned a DRB1 14 while DNA typing assigned DRB1 1117 The DRB1 14 serological reagents are monoclonal antibodies which are predicted to detect the epitope 31FHNQEEF37 based upon pattern analysis with DRB1 14 alleles This amino acid sequence is also shared with DRB1 1117 based on published amino acid sequences c The discord on DRB3 is based on one sample that was assigned a homozygous DRB1 08 by both serology and DNA typing but was assigned an additional DRB3 by serology DRB1 08 and DRB3 share amino a
3. a conserved region of the Human globin gene which is present in all human 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 HLA 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 HLA allele or allele group is visualized on the gel as an amplified DNA fragment between the internal control product band and the unincorporated primer band see Expected Values Gel Interpretation REAGENTS A Identification The Micro SSP DNA Typing Trays provide sequence specific oligonucleotide primers for amplification of HLA alleles and the human globin gene by the polymerase chain reaction PCR Pre optimized primers are presented dried in different wells of a 96 well 0 2 ml thin walled tube tray for PCR and are ready for the addition of DNA samples recombinant Taq polymerase and specially formulated dNTP buffer mix Micro SSP D mix Each typing tray includes a negative control reaction tube that detects the presence of the internal control PCR product generated by the Micro SSP
4. different methods Highlighted results indicate discordance between the two methods BIBLIOGRAPHY 1 Terasaki P I Bernoco F Park M S Ozturk G and Iwaki Y Microdroplet testing for HLA A B C and D antigens American Journal of Clinical Pathology 69 103 120 1978 2 Newton C R Graham A Heptinstall E Powell S J Summers C Kalsheker N Smith J C and Markham A F Analysis of any point mutation in DNA The amplification refractory mutation system ARMS Nucleic Acids Research 17 2503 2516 1989 3 Slater R D and Parham P Mutually exclusive public epitopes of HLA A B C Molecules Human Immunology 26 85 89 1989 4 Bodmer J Marsh S Albert E Bodmer W Bontrop R Charron D Dupont B Erlich H Mach B Mayr W Parham P Sasazuki T Schreuder G Strominger J Svejgaard A and Terasaki P Nomenclature for factors of the HLA system 1995 Human Immunology 43 149 164 1995 5 Terasaki P I Histocompatibility Testing 1980 UCLA Tissue Typing Laboratory Los Angeles California SSP DNAT PI EN 00 Rev 16 Page 7 of 8 6 Savage D A Bidwell J L Cullen C Bidwell E A and Middleton D Identification of HLA DRw52 associated antigens using HLA class II allogenotyping Tissue Antigens 32 278 285 1988 7 Sutton V R Kienzle B K and Knowles R W An altered splice site is found in the DRB4 gene that is not expressed in HLA DR7 Dw11 individuals Immunogenetics 29 317 322 1989 TROUBL
5. other DNA or PCR product Use new aliquots of all buffers reagent DNA extraction and PCR repeat DNA extraction and test SSP DNAT PI EN 00 Rev 16 Page 8 of 8 TRADEMARKS USED IN THIS DOCUMENT PRODUCT Micro SSP One Lambda Inc GeneAmp Applied Biosystems Fotodyne FOTO UV 21 FOTODYNE Incorporated FMC SeaKem FMC Corporation ARMS Zeneca Limited Gilson and Pipetman Rainin Instrument Co Inc Veriti Applied Biosystems PATENTS USED IN THIS DOCUMENT Notice to purchaser of licensed product The purchase price of this product includes limited non transferable rights under U S Patents 4 683 202 4 683 195 and 4 965 188 and their foreign counterparts owned by Roche Molecular Systems Inc and F Hoffmann LaRoche Ltd Roche to use only this amount of the product to practice the Polymerase Chain Reaction PCR Process described in said patents solely for the HLA Typing applications of the purchaser solely for organ bone marrow or tissue transplantation and explicitly excludes analysis of forensic evidence or parentage determination The right to use this product to perform and to offer commercial services for HLA Typing for organ or tissue transplantation using PCR including reporting the results of the purchaser s activities for a fee or other commercial consideration is also hereby granted Further information on purchasing licenses to practice PCR may be obtained by contacting in the United St
6. DNA Typing Tray The internal control PCR product amplified from the Human globin gene is the most likely contaminating PCR product due to its amplification in every well The amount of each primer is adjusted for optimal amplification of 100 ng of sample DNA when used in conjunction with the Micro SSP D mix the prescribed amount of recombinant Taq polymerase and the PCR reaction profile detailed in this document page 2 See the provided worksheet for specific alleles that can be amplified by each primer set under the specified PCR conditions of the assay For lot specific primer site locations refer to Primer Information document SSP DNAT PI EN 00 Rev 16 Page 2 of 8 B Warning or Caution 1 For In Vitro Diagnostic Use 2 Pipettes used for post PCR manipulations should not be used for pre PCR manipulations 3 Biohazard Warning The ethidium bromide used for staining of DNA is a potential carcinogen Always wear gloves when handling stained gels 4 Biohazard Warning All blood products should be treated as potentially infectious Source material from which this product was derived was found negative when tested in accordance with current FDA required tests No known test methods can offer assurance that products derived from human blood will not transmit infectious agents 5 Caution Wear UV blocking eye protection and do not view UV light source directly when viewing or photographing gels 6 See Material Safety Data Sheet for
7. ESHOOTING Problem Potential Causes Solutions No bands or faint bands DNA Not enough used Not pure enough A260 A280 not within 1 65 1 80 PCR inhibitor present e g EDTA above 0 5 mM Repeat test use amount indicated in the Micro SSP Product Reference Table Repeat DNA preparation check for A260 A280 within 1 65 1 80 then repeat test Repeat DNA preparation re suspend DNA in solution with EDTA lt 0 5 mM then repeat test Recombinant Taq polymerase Not enough used Enzyme degraded Repeat test use amount indicated in the Micro SSP Product Reference Table Repeat test use fresh tube of enzyme EtBr Not enough used Remake 1 X TBE with EtBr 0 5 g ml Repeat test Pressure Pad Pressure pad worn out Change pressure pad after 300 PCR runs False bands DNA Too much used Not pure enough A260 A280 not within 1 65 1 80 Contaminated other DNA or PCR product Repeat test use amount indicated in the Micro SSP Product Reference Table Repeat DNA preparation check for A260 A280 within 1 65 1 80 then repeat test Use new aliquots of all buffers reagents for DNA extraction and PCR repeat DNA extraction and test Recombinant Taq polymerase Too much used Repeat test use amount indicated in the Micro SSP Product Reference Table EtBr Too much used Remake 1 X TBE with EtBr 0 5 g ml Repeat test Bands in negative control Reagents Contaminated
8. SP Gel System OLI Cat MGS108 1 To set up Slide the locking pin on the base to the open position Insert the gel box into the base matching the color coded sides to assure proper orientation Lock the gel box into the base by sliding the locking pin into the locked position Use the leveling bubble and the three height adjustable legs to level the base 2 Orient and fully insert the 14 gel combs into the gel comb holder 3 To 100 ml of 1x Tris Borate EDTA buffer 1x TBE with 0 5 g ml ethidium bromide in a 500 ml glass bottle add 2 5 g electrophoresis grade agarose Heat until a homogeneous solution is formed SSP DNAT PI EN 00 Rev 16 Page 3 of 8 4 Add 30 ml of the gel solution to the gel box Make sure the agarose covers the entire surface 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 5 Remove the gel combs by lifting the gel comb holder while holding the base Add 10 ml of 1xTBE containing 0 5 g ml ethidium bromide evenly across the gel to fill every well C Gel Electrophoresis For Micro SSP Gel System OLI Cat MGS108 1 After completing the PCR reaction Orient the DNA primer set tray and gel box with the negative control well in the upper left hand corner Gently remove the tray seal without splashing the samples 2 Tr
9. SSP DNAT PI EN 00 Rev 16 Page 1 of 8 21001 Kittridge Street Canoga Park CA 91303 2801 USA Tel 1 818 702 0042 Fax 1 818 702 6904 www onelambda com P R O D U C T I N S E R T MICRO SSP HLA DNA TYPING TRAYS For In Vitro Diagnostic Use Important Refer to Micro SSP Product Reference Table Document ID MSSP_REF_TABLE_PI DOC for OLI product catalog numbers specific product components and requirements INTENDED USE DNA typing of Class I or Class II HLA alleles SUMMARY AND EXPLANATION Historically the established method for the determination of HLA antigens has been the lymphocytotoxicity test 1 However with the advent of PCR technologies DNA based tissue typing techniques have become routine in the laboratory For most DNA based methodologies the PCR process is used only as an amplification step to acquire the needed target DNA The HLA typing process then requires a post amplification step to discriminate between the different alleles e g RFLP SSOP reverse dot blot Unlike other PCR based methods the SSP methodology employed in the One Lambda Inc Micro SSP DNA typing test discriminates between the different alleles during the PCR process 2 This shortens the post amplification processing time to a simple gel electrophoresis detection step In contrast to the lymphocytotoxicity reaction scale 1 negative to 8 positive Micro SSP test results are either positive or negative This abolishes the need for co
10. ansfer each PCR reaction 10 l 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 Note The order of samples to match the worksheet is from left to right top to bottom 3 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 4 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 5 Orient the photograph with the negative control reaction in the upper left corner and mark the corresponding positive allele groups on the worksheet provided with the tray SPECIMEN COLLECTION AND PREPARATION A DNA can be purified from human leukocytes by any preferred method B 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 l with the A260 A280 ratio of 1 65 1 80 C Samples should not be re suspended in solutions containing chelating agents such as EDTA above 0 5 mM in concentration D DNA samples may be used immediately after isolation or stored at 20 C for extended period
11. ates the Director of Licensing at Roche Molecular Systems Inc 1145 Atlantic Avenue Alameda California 94501 and outside the United States the PCR Licensing Manager F Hoffmann La Roche Ltd Grenzacherstr 124 CH 4070 Basel Switzerland 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 Micro SSP DNA typing reagents are manufactured and distributed by One Lambda Inc 21001 Kittridge Street Canoga Park CA 91303 U S A Recombinant Taq polymerase is manufactured by F Hoffmann LaRoche EUROPEAN AUTHORIZED REPRESENTATIVE MDSS GmbH Schiffgraben 41 D 30175 Hannover Germany REVISION HISTORY Revision Date Revision Description 15 2009 02 Added the Veriti Thermal Cycler in the Instrument Requirements Section Separated Thermal Cycler into two words Template Update Inserted a Revision History Section 16 2009 07 Page 3 Materials Provided remove reference to Micro SSP Reference Table Move Taq Polymerase to Materials Required But Not Provided
12. ation of any well can void test results 2 A faster migrating positive typing band will be observed on the electrophoretic gel if a specific HLA 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 Match the pattern of positive wells with the information on the Micro SSP worksheet to obtain the HLA typing of the sample DNA 5 The presence of the internal control band and or the positive typing band in the negative control well voids all test results B Gel Interpretation Positive Reaction Negative Reaction Non Amplification Well Internal Control Band Positive Typing Band Primer Band The internal control band and broad unincorporated primer band serve as size markers Any visible band between the two size markers should be considered positive typing bands SPECIFIC PERFORMANCE CHARACTERISTICS A comparison of Micro SSP and serological HLA typing methodologies was performed to assess the accuracy of the Micro SSP HLA typing system In this comparison eighty one random whole blood samples were collected and analyzed for their HLA Class II typing using the One Lambda Inc HLA Class II Tissue Typing Tray DR72F and Micro SSP Generic HLA Class II DNA Typing Tray The results show a 96 78 81 concordance between the two methods The table on the following page represents the total number of times each specificity was assigned by the two
13. cid sequences which under the double dose effects of DRB1 08 homozygosity may generate false positive serological reactions 6 d The false negative discord on DRB4 is based on one sample in which only DNA typing assigned a DRB4 Assignments by both serology and DNA typing show that this sample contains the DR7 DQ9 haplotype which is known to lack expression of the DRB4 allele at the protein level 7 Note Lot specific performance data is available on request SSP DNAT PI EN 00 Rev 16 Page 6 of 8 DQB1 0302 0304 11 11 DQB1 0303 3 3 DQB1 04 11 11 LIMITATIONS OF THE PROCEDURE A PCR SSP is a dynamic process requiring highly controlled conditions to insure discriminatory amplification The procedure provided in this product must be strictly followed B 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 C All instruments e g PCR machine pipetting devices must be calibrated according to the manufacturer s recommendations For lot specific information refer to the Resolution Limitations document D For lot specific information refer to the Resolution Limitations document EXPECTED VALUES 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 amplific
14. detailed information C Instructions for Use See Directions for Use D Storage Instructions Store reagents at temperature indicated on package Use before printed expiration date E Purification or Treatment Required for Use See Directions for Use F Instability Indications 1 Do not use primer set trays with cracks in the tubes or on the lips cracks may cause evaporative losses 2 If salts have precipitated out of the solution in the D mix aliquots during shipping or storage re dissolve them by extended vortexing at room temperature 20 to 25 C 3 D mix aliquots upon thawing at room temperature 20 to 25 C should be pink to light purple in color Any D mix aliquot without the specified coloration should be considered unusable INSTRUMENT REQUIREMENTS A Programming the Thermal cycler 96 Well GeneAmp PCR System 9600 9700 or Veriti 96 Well Thermal Cycler Applied Biosystems Set ramp speed to 9600 for 96 Well GeneAmp PCR System 9700 Set ramp speed to 9600 Emulation Mode for Veriti 96 Well Thermal Cycler Program your thermal cycler before starting the Step by Step Procedure below One Lambda PCR Program OLI 1 of Cycles Step Temp C Time 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 thermal cycler information refer to the manufacturer s user manual B 2 5 Agarose Gel Preparation For Micro S
15. ent evaporative loss during PCR 11 Place the Micro SSP primer set tray in the thermal cycler 12 Place a pressure pad on top of the tray with the textured side up before closing the thermal cycler lid See Micro SSP Pressure Pad Protocol for instructions on specific pad to be used with your thermal cycler 13 Enter your Micro SSP PCR program number Specify a 10 l reaction volume option 14 Start the PCR program The program takes approximately 1 hour and 16 minutes The last step will hold the sample at 4 C until the run is stopped 15 Remove the primer set tray from the thermal cycler Gently remove the tray seal without splashing the samples Or store the samples in the primer set tray at 20 C or below for later gel electrophoresis 16 Transfer each PCR reaction 10 l in sequence to a 2 5 agarose gel in the Micro SSP Gel System OLI Cat MGS108 Use of an 8 or 12 channel Pipetman or of the 96 Well Transfer Device OLI Cat TRNDV96 is highly recommended Note Be sure to transfer all samples in the proper sequence orienting the primer set tray with the negative control reaction well in the upper left corner The order of samples to match the worksheet is from left to right top to bottom No addition of electrophoresis dye is necessary 17 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
16. mplicated interpretation of results In addition single nucleotide changes can be discriminatory in PCR SSP while cross reacting groups CREGs provide major challenges to serological typing 3 Finally due to the synthetic nature of DNA typing reagents e g oligonucleotide primers stability has been greatly improved and lot to lot variance has been reduced PRINCIPLE S 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 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 a specific amplified DNA fragment Since amplification during the PCR reaction 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
17. s of time over 1 year with no adverse effects on results E DNA samples should be shipped at 4 C or below to preserve their integrity during transport PROCEDURE A Materials Provided 1 Micro SSP DNA primer set trays 2 Pre aliquoted D mix tubes appropriate number for test 3 Tray seals appropriate number for test B Materials Required But Not Provided Recombinant Taq polymerase 5 units l Pipetting devices such as Gilson P 20 Gilson P200 Pipetman Disposable pipette tips Vortex mixer Microcentrifuge PCR tray microtube storage rack and cover Robbins Scientific Cat 1044 39 5 SSP DNAT PI EN 00 Rev 16 Page 4 of 8 Pressure pad for use with tray OLI Cat SSPPAD SSPPAD384 SSPPADGRY Use only the pressure pad specifically for your thermal cycler See Micro SSP Pressure Pad Protocol PCR_PAD_INS DOC Note The pressure pad is good for a maximum of 300 PCR runs Please order a new pad if the surface of the pad that contacts the tray seal is no longer smooth Thermal cycler for PCR with 96 well format and tray retainer for 0 2 ml thin walled reaction tubes Hot plate or microwave oven for heating agarose solutions Electrophoresis apparatus power supply 150V minimum capacity UV transilluminator Example Fotodyne FOTO UV 21 Photographic or image documentation system 1x TBE buffer 89mM Tris borate 2 mM disodium EDTA pH 8 0 with
18. ted below for pipetting Taq polymerase Note Taq polymerase is very viscous and special care must be taken in the aliquoting process Failure to follow the steps described below may result in reagent loss 1 Pipette slowly using a calibrated Gilson Pipetman A P10 Pipetman is recommended for increased accuracy 2 The pipette tip should just pass the surface layer of the Taq Warning Do not immerse the pipette tip in the Taq 3 Carefully wipe excess Taq from pipette tip on the rim of the vial D Stepwise Procedure 1 Remove from the indicated storage temperature the volume tube of the Micro SSP D Mix that matches the selected Micro SSP primer set tray the primer set tray s and the appropriate number of DNA samples Thaw at room temperature 20 to 25 C Note You may cut tray and tray seal into the number of tests needed for a single work session Immediately return the un used portions to the appropriate storage temperature Vortex DNA samples to mix Place the primer set tray in a PCR Tray Microtube Storage Rack Robbins Scientific Cat 1044 39 5 and remove the tray label 2 Remove recombinant Taq polymerase from freezer and keep on ice until ready to use 3 Using a Pipetman or equivalent add 1 l of DNA diluent to the negative control reaction tube on the primer set tray 4 Using a Pipetman or equivalent add recombinant Taq polymerase 5 units l to the Micro SSP D mi
19. x tube See Micro SSP Product Reference Table for amount SSP DNAT PI EN 00 Rev 16 Page 5 of 8 5 Cap tube and vortex for 5 seconds Pulse spin the Micro SSP D mix tube in a microcentrifuge to bring all liquid down from sides of the tube 6 Using a P20 Pipetman or equivalent pipette 9 l of the Micro SSP D mix to the negative control reaction tube 7 Using a Pipetman or equivalent add the DNA sample to the Micro SSP D mix tube See Micro SSP Product Reference Table for amount 8 Cap tube and vortex for 5 seconds Pulse spin the Micro SSP D mix tube in a microcentrifuge 9 Using a P20 Pipetman or an electronic Pipetman aliquot 10 l of the sample reaction mixture from the Micro SSP D mix tube into each reaction tube except the negative control reaction tube of the Micro SSP primer set tray Important Be sure to apply the sample above the primers dried at the bottom of each reaction tube to avoid cross contamination between tubes Touch the inside wall of the tube with the pipette tip to allow the sample to slide down to the bottom of the tube Check that all samples have dropped to the bottom of each tube If not tap the tray gently on the bench top so that all samples settle at the bottom of the tube before you begin PCR 10 Cover the reaction tubes with the tray seal provided Check that all reaction tubes are completely covered by the tray seal to assure complete closure and to prev
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