User Manual Protrans HLA SSP Kits
Contents
1. PROTRANS HLA class ys HLA class Il Positive reaction Gel pocket Gel pocket Specific band Control band gt 100 900bp 440bp Control band SSS Specific band 89 bp lt 260bp Primer cloud Primer cloud 1 If weak bands of incorrect size are present disregard them if the overall strength and clarity of the amplification is good 2 Unused primer will form a diffuse band below 50 base pair 3 Occasionally the formation of primer dimers lt 80 bp can be observed The primer dimers do not invalidate the test A wrong interpretation of the result can be avoided by checking the approximate product size against the correct product size of the PROTRANS Primer Mix Table see package insert 4 Some of the primer mixes of HLA A mixes 5 16 19 and 24 and of HLA B mixes 5 9 43 and 47 will give short PCR products lt 200 bp These PCR products might be difficult to distinguish from the 89 bp control PCR product In general these specific products will give a much stronger signal than that of the controls and will not have migrated as far into the gel as the control bands If you are not sure whether the strong signal is due to a specific or a generic PCR product you might let the gel run for an additional 15 minutes at a lower voltage This way the specific band will be separated from the control band and you will be able to clearly see a double signal at this position a very strong specific and a weaker2. le negatif Pour le diagnostic invitro Conserver P remption Introduction 1 1 Intended Use The Protrans HLA Sequence Specific Primer SSP Kits are intended for the determination of HLA class or class II alleles based on the PCR SSP method 1 2 Summary and Explanation The HLA system is a complex co dominantly inherited system of antigens which plays an important role in the immune system by enabling it to distinguish self from non self In organ transplantation HLA compatibility between donor and recipient is one of the major determinants of transplant outcome For this reason the determination of the individual combinations of HLA antigens is used as a basis for the selection of donors and recipients New dimensions have been opened up to modern diagnostics by the development of DNA based test methods HLA antigens partly differ from each other only by single amino acids within the polypeptide chain Recognition of these largely identical structures by serological means is almost impossible for this reason the resolution capacity of the method is limited As the DNA sequences of most important HLA alleles are now known variations in sequences can be identified at the DNA level with the help of synthetic oligo nucleotides Utilizing amplifications of genomic DNA PCR Polymerase Chain Reaction together with specific primer pairs SSP Sequence Specific Primer it is possible to identify a large number of HLA alleles by mole
3. 1 Gel electrophoresis The PCR products are identified using agarose gel electrophoresis followed by the detection of the DNA bands in UV light 8 2 Performing gel electrophoresis A 2 solution of agarose is prepared by boiling 4g of agarose in 200ml of 1x TAE using a magnetic stirring hotplate or microwave oven until the solution becomes clear Allow the solution to cool to below 60 C before adding 10ul of ethidium bromide solution TN voir bromide solution 10mg ml Dissolve 100mg of ethidium bromide in 10ml of distilled water Store at 2 8 C protected from light Caution Ethidium bromide is mutagenic and toxic Always wear protective gloves when working with ethidium bomide also in diluted form In case of contact with the skin wash off immediately with copious amounts of water Place the PROTRANS UV Gel Tray on a horizontal surface Fill the agarose solution into the PROTRANS UV Gel Tray avoiding air bubbles and place the PROTRANS Gel Combs 4 combs with x 4x25 slots each containing 10ul into the gel The distance of the combs teeth correspond to that of a standard 8 channel pipette which allows rapid load of the samples onto the gel After polymerization about 30 60 minutes at room temperature remove the combs and place the gel into the gel chamber that has been filled with 1x TAE buffer The level of electrophoresis buffer should be 2 3 mm above the gel surface and completely cover the gel Carefully remove the cap
4. DQA1 high R 70 Y 140 1 6 ul A3 6 9 12 50 ul 7 7 Number and Positions of Primer Mixes of PROTRANS Cyclerplate System REF Protrans Cyclerplate Number of Position of Position NC System Primer Mixes 1 Primer Mix 200 070 HLA A 4x 24 H1 4 7 10 200 080 HLA B 2x 48 H1 7 200 090 HLA C 4x 24 H1 4 7 10 A3 6 9 12 200 030 HLA DRB1 4x 24 H1 4 7 10 A 3 6 9 12 200 040 HLA DQB1 low 6x 14 H1 3 5 7 9 11 C 2 4 6 8 10 12 200 020 HLA A B DRB1 96 A H1 B H4 DRB1 H10 A12 200 010 HLA A B C 96 A H1 B H4 Cw H10 A12 200 011 HLA A B 73 A H1 B H4 H10 200 050 HLA DRB1 DQB1 2x 38 DRB1 H1 H7 DQB1 H4 H10 __ A3 C5 A9 C11 200 048 HLA DQB1 high 2x 47 H1 H7 B6 B12 200 049 HLA DQA1 high 4x 24 H1 H4 H7 H10 A3 A6 A9 A12 User Manual PROTRANS HLA SSP Cyclerplate and Domino System Version 2 4 Rev 2009 10 20 page 9 of 16 7 8 PROTRANS Domino System REF Article Buffer Buffer 201 071 HLA DRB1 01 R Y 201 072 HLA DRB1 03 R Y 201 070 HLA DRB1 04 R Y 201 077 HLA DRB1 07 09 R Y 201 073 HLA DRB1 08 12 R Y 201 074 HLA DRB1 11 R Y 201 075 HLA DRB1 13 R Y 201 076 HLA DRB1 14 R Y 201 078 HLA DRB1 15 R Y 201 079 HLA DRB1 16 R Y 201 080 HLA DRB1 15 16 R Y 7 9 Volumes of PROTRANS Buffers Taq Polyme
5. Vortex Mastermix well When using a negative control pipette 10 ul of this Mastermix without the DNA into the appropriate well See Tables 7 6 7 7 and 7 9 7 10 Add to each Mastermix the appropriate volume of DNA recommended for the test See Tables 7 6 and 7 9 7 2 5 Vortex Mastermix well 7 2 3 7 2 4 7 3 Dispensing the Master Mix in the Protrans PCR Cyclerplates or PCR Domino Strips Dispense 10 ul of the Mastermix into each well of the PCR Cyclerplate or PCR Domino Strip but not into the negative control When pipetting at the upper edge of each well the drop will run down to the bottom The bottom of the wells must not be touched with the tip 7 3 1 7 4 Capping the Protrans PCR Cyclerplates or PCR Domino Strips Ensure that the Mastermix is at the bottom of all wells by either centrifugation or gentle tapping the 7 4 1 Cyclerplate or Domino Strip onto the work bench Visual control ensures that the Mastermix is at the bottom of each well Seal Cylerplates or Domino Strips carefully using the caps provided or with re usable PROTRANS Tae 24 96 well Coverplates Ensure caps completely seal the wells to prevent evaporation Place the Cyclerplate or Domino Strips into Thermocycler and start amplification 743 Final volume in each well is 10 ul If unable to start amplification immediately after PCR set up the PCR Cyclerplates or Domino Strips must be stored at 2 8 C Start ampli
6. in combination Buffer D and Buffer Y PROTRANS Cyclerplates and PROTRANS Domino Strips contain pre pipetted dried specific Primer Mixes oligonucleotides The positions and specificities of the Primer Mixes are LOT specific and described in the Kit Inserts Reaction Protocol Primer MixTable Amplification Table and Typing Table Number of Primer Mixes of each Testkit in the PROTRANS Cyclerplate System refer to Table 7 7 Number of Primer Mixes of each Testkit in the PROTRANS Domino System refer to Table 7 10 The PROTRANS Cyclerplates are fit into each other and packed in zip lock pouches Each pouch contains 10 or 20 Cyclerplates The pouch and the upper Cyclerplate are marked with a label Name of the product specificities of the pre pipetted Primer Mixes LOT number storage temperature and expiration date are written on the label Name of the product and LOT number are printed on each PROTRANS Cyclerplate Each well of the PROTRANS Cyclerplate is marked with a digit letter combination from A1 to H12 Digits are visible on the top letters on the left edge of the Cyclerplate Primer Mix 1 of each Testkit is located at position H1 For better orientation the lower edge of the Cyclerplate has been marked with a black line 96 wells 16 24 32 40 48 56 64 72 80 88 96 15 23 31 39 47 55 63 71 79 87 95 14 22 30 38 46 54 62 70 78 86 94 13 21 29 37
7. of the Procedure 10 1 Intensity of positive bands will vary due to the quality and amount of the PCR product Quality of the PCR product will directly affect the intensity of the specific and the control bands visualized through the UV Transilluminator In case of missing internal control bands and no accounted alleles the test should be repeated 2 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 test results 3 Performance of the test can only be guaranteed if enclosed instructions are strictly adhered to 4 The SSP HLA Typing tests should only be used for initial HLA typing Other clinical and diagnostics findings should be used in addition when determining suitability for transplant 5 Use of the PROTRANS SSP HLA typing kits cannot resolve all combinations Quality Control Each manufactured LOT is checked against a panel of DNA samples representative of specific detection by the primer See certificates of Analysis for each LOT All PROTRANS SSP kits are produced on the basis of the quality standards EN ISO 9001 2008 EN ISO 13485 2003 AC 2007 and attachment IV of the IVDD 9879 EG User Manual PROTRANS HLA SSP Cyclerplate and Domino System Version 2 4 Rev 2009 10 20 page 13 of 16 11 Troubleshooting Problem Possible Cause Solution No PCR products Multiple thawing and freezing of the Mastermi
8. 1997 Arnett K L and Parham P HLA Class nucleotide sequences 1995 Tissue Antigens V 46 217 257 1995 Olerup O and Zetterquist H HLA DR typing by PCR amplification with sequence specific primer PCR SSP in 2 hours An alternative to serological DR typing in clinical practice including donor recipient matching in cadaveric transplantations Tissue Antigens V 39 225 235 1992 Specific enzymatic amplification of DNA in vitro The Polymerase Chain Reaction Mullis et all Cold Spring Harbour Symposia on quantitive biology Vol 51 No 1 p 263 273 1986 Bunce O Neill Barnado Krausa Browning Morris Welsh 1995 Phototyping comprehensive DNA Typing for HLA A B C DRB1 DRB3 DRB4 DRB5 amp DQB1 by PCR with 144 mixes utilizing sequence specific primer PCR SSP Tissue Antigens 46 355 367 Blasczyk Hahn Wehling Huhn Salama 1995 Complete subtyping of the HLA A locus by sequence specific amplification followed by direct sequencing or single strand conformation polymorphism analysis Tissue Antigens 46 86 95 OMOT ONS medizinische diagnostische produkte gmbh User Manual PROTRANS HLA SSP Cyclerplate and Domino System Version 2 4 Rev 2009 10 20 page 16 of 16
9. 45 53 61 69 77 85 93 12 20 28 36 44 52 60 68 76 84 92 11 19 27 35 43 51 59 67 75 83 91 10 18 26 34 42 50 58 66 74 82 90 9 17 25 33 41 49 57 6 73 81 89 eo DT INI AJA OQ N oo The PROTRANS Domino Strips are fit into each other and packed in zip lock pouches Each pouch contains 12 24 or 30 Domino Strips The pouch is marked with a label On the label Name of the product specificities of the pre pipetted Primer Mixes LOT number storage temperature and expiration date are written PROTRANS Domino System Position Primer Mix 1 Domino Strip 8 wells 16 wells 24 wells 32 wells 8 8 16 8 16 24 8 16 24 32 7 7 15 7 15 23 7 15 23 31 6 6 14 6 14 22 6 14 22 30 5 5 13 5 13 21 5 13 21 29 4 4 12 4 12 20 4 12 20 28 3 3 11 3 11 19 3 11 19 27 2 2 10 2 10 18 2 10 18 26 1 1 9 1 9 17 1 9 17 25 ae 2s js 5 3 5 2 User Manual PROTRANS HLA SSP Cyclerplate and Domino System Version 2 4 Rev 2009 10 20 page 4 of 16 2 2 Warning and Precautions IVD Reagents only for In Vitro Diagnostic use A The PROTRANS Testkits must be performed by well trained and authorised laboratory technicians All reagents shou
10. CR amplification This amplification generally always occurs i e both in presence or in absence of an allele or group specific PCR fragment The control band can therefore generally be seen in all PCR reactions In the presence of an allele specific PCR product the control band can appear weaker or is completely missing This is not a limitation of the method as the specific band provides a check on the success of the PCR amplification The composition of the primer mixes permits positive identification of the HLA characteristics The interpretation is based on whether a specific band is present or not The size of the amplified DNA fragment does not need to be taken into consideration when evaluating the test nevertheless it might be helpful for the test interpretation For evaluation the pattern of the specific bands is transferred to the LOT specific Protrans Reaction Protocol supplied and the typing result read off with the aid of the reaction pattern For the interpretation the Primer Mix tables and the Amplification tables are very helpful Additionally the Score Program www ihwg org can be used for detailed result interpretation If the testkit contains a negative control any band in this amplification is evidence of contamination The results of the test would be invalid and the test must be repeated User Manual PROTRANS HLA SSP Cyclerplate and Domino System Version 2 4 Rev 2009 10 20 page 11 of 16 8 5 Gel Interpretation
11. Cli User Manual Protrans HLA SSP Kits Molecular genetic DNA Typing SSP technique PROTRANS PROTRANS Cyclerplate System Domino System REF Article REF Article 200 070 HLA A 201 071 HLA DRB1 01 200 080 HLA B 201 072 HLA DRB1 03 200 030 HLA DRB1 VD 201 070 HLA DRB1 04 200 020 HLA A B DRB1 201 077 HLA DRB1 07 09 200 010 HLA A B C ce 201 073 HLA DRB1 08 12 VD 200 011 HLA A B 201 074 HLA DRB1 11 200 050 HLA DRB1 DQB1 201 075 HLA DRB1 13 ce 200 090 HLA C 201 076 HLA DRB1 14 200 040 HLA DQB1 low 201 078 HLA DRB1 15 201 201 Negative Control HLA IVD 201 079 HLA DRB1 16 200 048 HLA DQB1 high CE 201 080 HLA DRB1 15 16 200 049 HLA DQA1 high IVD For In Vitro Diagnostic use Buffer D R and Y Cyclerplates and Domino Strips See label on the Testkit User Manual Warning and Precautions 20 C 2 8 C Protrans medizinische diagnostische Produkte GmbH D 68766 Hockenheim Ketschau 2 Tel 0049 0 6205 2929910 Fax 0049 0 6205 2929920 www protrans info mail protrans info User Manual PROTRANS HLA SSP Cyclerplate and Domino System Version 2 4 Rev 2009 10 20 page 1 of 16 Content xo Q Content Quality and Certification License ARMS
12. PCR 11 8 1 Gel electrophoresis 11 8 2 Performing Gel electrophoresis 11 8 3 Result Interpretation and result documentation 11 8 4 Result Interpretation 11 8 5 Gel Interpretation 12 9 Limitations of the Procedure 13 10 Quality Control 13 11 Trouble Shooting 14 12 Literature 16 User Manual PROTRANS HLA SSP Cyclerplate and Domino System Version 2 4 Rev 2009 10 20 page 2 of 16 Quality and Certification We declare under our sole responsibility that the mentioned products are in compliance with the essential requirements of the Directive IVDD 98 79 EG and EN ISO 13485 2003 and EN ISO 9001 2000 on design production and sales of HLA tissue typing products and are marked with the C symbol License ARMS technology With the Protrans SSP Typing Kits the ARMS technology is used The Products are sold under license of BTG International Limited UK and DxS Ltd UK ARMS is the subject of worldwide patent property and is a trademark of the AstraZeneca group of companies Symbols Country REF NC IVD DE Artikelnummer Negativkontrolle in vitro Diagnostik Lagern bei Haltbar bis GB Article number negative controll For in vitro diagnostic use Store at Expiration Date ES Artikulo n mero control negativo S lo para el diagn stico in vitro Almacenar Caducidad IT Codice prodotto controllo negativo Solo per la diagnostica in vitro Conservare a Scadenza FR Num ro de I articl contr
13. al materials reagents and equipment required but not supplied All reagents and equipment other than recommended requires validation by the user 5 2 1 UV spectrophotometer for photometric DNA measurement e g Lambda Scan 200 MWG www THE MWG com PC photometer program KC 4 Bio Tek Instruments Inc www biotek com Use of other UV spectral photometers must be validated by the user 5 2 2 DNA Taq Polymerase The following enzymes are validated for use with the PROTRANS SSP Kits e AmpliTaq Perkin Elmer 5U ul Cat No N8010060 e MBI Fermentas DNA Taq Polymerase 5U ul Cat No EP0402 Use of other DNA Taq Polymerase enzymes must be validated by the user 5 2 3 Thermocycler The following Thermocyclers with 96 well block and heated lid are validated for use with the PROTRANS SSP Kits e PE 2700 Applied Biosystems www appliedbiosystems com production PE 9600 Applied Biosystems stopped e PTC 100 PTC 200 MJ Research Inc www mir com Thermocyclers other than the recommended have to be user validated Thermocyclers which have no adjustable pressure plate require an adaptor mat in order to guarantee optimal heat transfer from the heat cover to the PCR tubes 5 2 4 Pipettes e Adjustable pipettes for volumes o 1 104 o 10 100yl o 100 1000 e Eppendorf Multipette Type 4720 e 8 channel pipette e g 5 50ul adjustable pipetting volume Finnpipette ThermoLabsystems Cat No 4510020 5 2 5 Disposables e Suitable Filtertips specific f
14. be kept closed in their original package Reseal the ziplock to prevent moisture accumulation during storage User Manual PROTRANS HLA SSP Cyclerplate and Domino System Version 2 4 Rev 2009 10 20 page 5 of 16 3 Instrument requirements 3 1 Programming the Thermocycler Thermal Cycling Program For optimal results it is important to obtain rapid ramp times 1 2 5 C s and precise temperature control The following thermal cycler profile is optimized and validated with the thermocyclers given in 5 2 3 for use with the PROTRANS SSP Cyclerplate System and Domino System The final volume of the amplification mix is 10 pl Initial denaturation 94 C 2 min Hold Denaturation Annealing and Extension 1O Eyles Denaturation Annealing 20 cycles Extension Hold 4 C 15 min Hold See 5 2 3 list of validated Thermocyclers with 96 well block and heated lid 4 Specimen collection and preparation 4 1 DNA Isolation 4 2 Genomic DNA may be obtained from all nucleated cells Starting materials are EDTA or citrate blood buffy coat or cell suspensions A vast range of various protocols exist for the isolation of DNA For PCR SSP testing only those methods which provide DNA of adequate quality and quantity should be considered The Protrans DNA Extraction Kit PROTRANS DNA Box 500 5000 provides DNA of high stability and quality All DNA extraction methods must be validated before routine use The concentration of t
15. cular test methods 1 3 Principle of the Test User Manual Protrans HLA SSP Kits are intend to determine HLA class or class II alleles based on a PCR method using sequence specific primer PCR SSP The principle of the SSP method is to generate an amplificate only when the sequence of a primer is perfectly complementary to the target sequence of a DNA sample On the other hand non complementary primer do not bind to the DNA and for this reason no amplification takes place Using agarose gel electrophoresis the amplified DNA can be determined Successful amplification will generate a DNA fragment of defined length which appears as a band in the gel If no amplification takes place no specific band is seen The composition of the primer mixes in each well of the Cyclerplates and Domino Strips permits positive identification of different specific alleles of each HLA locus The Protrans SSP kits are in Vitro Diagnostic Test systems for the diagnosis of the immune system of organ donors and recipients and of patients receiving blood component substitution therapy PROTRANS HLA SSP Cyclerplate and Domino System Version 2 4 Rev 2009 10 20 page 3 of 16 2 Reagents 2 1 Contents of the Protrans SSP Kits Component 1 Cyclerplates or Domino Strips Oligonucleotides dried 2 8 C Component 2 HLA class 1 Buffer D and Buffer Y HLA class 2 Buffer R and Buffer Y Master Mix for Amplification 20 C HLA class 1 HLA class 2
16. epeat the test Specific bands occasionally missing Inefficient Thermocycler e g block defect Control of Thermocycler by Protrans Cycler Check REF 200 100 Repeat test with validated Thermocycler see 5 2 3 The described program applies to the recom mended thermal cyclers Other thermal cycler as recommended have to be user validated Contact of the PCR plate to the block of the Thermocycler is not correct The provided rack from PE sample holder must not be used in combination with the 96 well PCR Cyclerplates DNA not evenly re suspended in original diluent or in Mastermix Vortex DNA sample and Mastermix thoroughly before PCR set up and repeat test If two alleles can be positively identified no action need to be taken In all other cases it is recommended to repeat the test Wrong loading of the agarose gel Control of gel and gel lanes concerning the loaded primer mixes False positive bands Inefficient Thermocycler e g block defect Control of Thermocycler by Protrans Cycler Check REF 200 100 Repeat test with validated Thermocycler see 5 2 3 The described program applies to the recom mended thermal cyclers Other thermal cycler as recommended have to be user validated Excess of DNA Repeat test with adequate amount of DNA Concentration 100ng ul Excess of DNA Taq Polymerase Repeat test with adequate amount of Taq Validate each LOT
17. fication within 2 hours User Manual PROTRANS HLA SSP Cyclerplate and Domino System Version 2 4 Rev 2009 10 20 page 8 of 16 7 5 PROTRANS Cyclerplate System REF Article Buffer Buffer 200 070 HLA A D Y 200 080 HLA B D Y 200 090 HLA C D Y 200 030 HLA DRB1 R Y 200 040 HLA DQB1 low R Y 200 020 HLA A B DRB1 D Y 200 010 HLA A B C D Y 200 011 HLA A B D Y 201 201 Negative Control HLA DorR Y 200 050 HLA DRB1 DQB1 R Y 200 048 HLA DQB1 high R Y 200 049 HLA DQA1 high R Y 7 6 Volumes of PROTRANS Buffers and Taq Polymerase for the Master Mix RER even nan Burreniul Buffer 4 Boeli s0100 200 070 HLA A D 70 Y 140 1 6ylj 50 ul 200 080 HLA B D 140 Y 280 3 3 yll 100 ul 200 090 HLA C D 70 Y 140 1 6 ul A3 6 9 12 50 ul 200 030 HLA DRB1 R 70 Y 140 1 6 ul A3 6 9 12 50 ul 200 040 HLA DQB1 low R 46 Y 94 1 1 ul C2 4 6 8 10 12 34 ul 200 020 HLA A B DRB1 D 280 Y 560 6 5 ul A12 200 ul 200 010 HLA A B C D 280 Y 560 6 5 ul A12 200 ul 200 011 HLA A B D 210 Y 420 5 0 ul H10 150 ul 201 201 Negative Control HLA DorR Y Volume as indicated for each REF 200 050 HLA DRB1 DQB1 R 115 Y 230 2 8 ul A3 9C 5 11 85 ul 200 048 HLA DQB1 high R 140 Y 280 3 3 ul B 6 12 100 ul 200 049 HLA
18. he DNA should be adjusted to 50 100 ng pl The ratio purity A260 A280 should be 1 6 1 8 Concentration and purity of the DNA is of decisive importance for optimal test results DNA sample may be used immediately after isolation or stored at 20 C or below for extended period of time over one year with no adverse effects on the results For storage and stability information of isolated DNA please refer to the technical information provided by the DNA extraction test kit manufacturer Use either EDTA or Citrate blood samples for DNA extraction Do not use heparinized blood samples Heparin inhibits the PCR Non acceptable specimens Contamination of the DNA by PCR inhibitors such as haemoglobin heparin ethanol ficoll separated specimens buffy coat cell suspensions etc can result in serious interference with the PCR reaction For this reason heparin blood is not acceptable as a starting material for DNA isolation If the patient is on heparin therapy use an alternative source of DNA Avoid the use of lipemic or haemolysed specimens The use of specimens collected without anticoagulant or frozen thawed multiple times is not recommended since these conditions may not provide sufficient quantity or quality of DNA for testing User Manual PROTRANS HLA SSP Cyclerplate and Domino System Version 2 4 Rev 2009 10 20 page 6 of 16 5 Materials 5 1 Materials Provided See 2 1 contents of the PROTRANS SSFP kits 5 2 Addition
19. ld be handled in accordance to good laboratory practice using appropriate precautions In addition handle all patient samples as potentially infectious Do not pipette by mouth All used PCR Cyclerplates and Domino Strips should be treated as potentially infectious and should be destroyed according to the valid national guidelines Do not use reagents which are expired See expiration date printed on the label Pre PCR and Post PCR rooms must be strictly separated Use separate pipettes in the Pre PCR area and in the Post PCR area Ethidium bromide used for staining of DNA is a potential carcinogen Always wear protective gloves when handling Ethidium bromide and stained gels Waste management according to national guidelines Wear UV blocking eye protection and avoid direct UV light when viewing or photographing gels See Material Safety Data Sheet MSDS for detailed information Available from Protrans 2 3 Storage and shelf life Kit Components Ingredients i Cyclerplates 96 well PCR plates Primer DNA Oligo Nucleotides Domino Strips Cresol Red 2 8 C See label 32 24 16 or 8 well PCR Strips Ammonium Sulfate Tris Buffer MgCl2 Glycerol A Buffer D R Cresol Red dNTPs Deoxyribonucleotides 20 C _ See label Buffer Y Watery solution 20 C _ See label Once the test kits have been opened the remaining unused Cyclerplates or Domino Strips must
20. of DNA Taq Polymerase before using it routinely Extensive delay between PCR set up and start of thermal cycling Start amplification directly after PCR set up or store pipetted Cyclerplate at 2 8 C until start of amplification max for 2 hours Mis interpretation of primer dimer as specific amplification Check correct band size DNA contaminated with other DNA or PCR product Separate strictly pre PCR area from post PCR area Perform wipe test Overall fuzzy bands smeared lanes Gel is too thin due to excess evaporation while heating Compensate for lost volume by adding water Agarose not completely dissolved Boil for an additional 30 seconds after melting Overheating gel too high voltage Lower voltage Heavy streaking in random wells can be caused by uneven suspensions of DNA Using an 8 channel pipettor mix the PCR product up and down twice before loading Rapid release of amplified product during gel loading can cause product to float out of well Use slow steady pipetting when loading gel Occasional faint lanes Product floated out of gel slot Pipette tip needs to be properly aligned with the slots of the agarose gel Gel picture too dark Wrong amount of Ethidium bromide used Use 5ul Ethidium bromide 10mg ml for each 100ml gel solution Gel tray not UV transparent Remove gel from tray before viewing Use PROTRANS Electropho
21. ontrol of thermocycler by Protrans Cycler Check REF 200 100 Repeat test with validated Thermocycler see 5 2 3 The described program applies to the recom mended thermal cyclers Other thermal cycler as recommended have to be user validated Contact of the PCR plate to the block of the thermocycler is not correct The provided rack from PE sample holder must not be used in combination with the 96 well PCR Cyclerplates PCR inhibitors heparin ficoll ethanol in the genomic DNA Impure DNA see 4 2 Repeat test with re extracted DNA from appropriate sample EDTA or citrate blood Use approx 100 ng ul DNA After washing the DNA pellet with wash buffer including ethanol allow sample to dry thoroughly DNA concentration used was too high low Repeat test with correct DNA concentration Degraded DNA smears in gel lanes Repeat test with a new re extracted DNA sample Lack of DNA Taq Polymerase activity Repeat test with a known reference DNA to verify activity of Taq Polymerase Non specific bands Inefficient Thermocycler e g block defect Control of Thermocycler by Protrans Cycler Check REF 200 100 Repeat test with validated Thermocycler see 5 2 3 The described program applies to the recom mended thermal cyclers Other thermal cycler as recommended have to be user validated Incorrect thermocycler program used Repeat test with correct amplification p
22. or each pipettes e 1 5 ml polypropylene reaction tubes e g Eppendorf Safe Lock Cat No 0030 120 86 e 0 5 ml Combitips for Eppendorf Multipette Type 4720 5 2 6 Gel Electrophoresis Agarose for molecular biology TAE Buffer 1x electrophoresis buffer e TAE Tris buffer conc Acetic Acid CH3COOH 0 5 M NA2 EDTA ph 8 0 Distilled water dH20 Ethidium bromide solution 10mg ml Caution Ethidium bromide is mutagenic see 2 2 Magnetic stirrer with hotplate or microwave oven DNA molecular weight marker 50 1000 bp ladder Power Supply PROTRANS Electrophoresis System PROTRANS Electrophoresis Unit or Gel Check REF 210 000 210 001 Gel documentation System e Polaroid camera with UV Filter and Polaroid film type 667 e Gel documentation system e Transilluminator 312nm UV light User Manual PROTRANS HLA SSP Cyclerplate and Domino System Version 2 4 Rev 2009 10 20 page 7 of 16 6 PCR Polymerase Chain Reaction 6 1 Precautionary measures PCR is an extremely sensitive method which can efficiently amplify even the smallest amounts of DNA It follows from this that even traces of contaminating DNA in a sample can be amplified in the PCR reaction and falsifies the test results One particular source of contamination is amplified DNA coming into contact with samples which are still to be amplified To avoid contamination with amplified material it is recommended that the work areas be strictly separated as follows 6 2 P
23. rase and DNA for the Master Mix REF eee ee Burreripl Beery a in Ba A sto 201 071 HLA DRB1 01 R 46 Y 94 1 1 ul 13 34 ul 201 072 HLA DRB1 03 R 70 Y 140 1 6 ul 20 50 ul 201 070 HLA DRB1 04 R 95 Y 190 2 2 ul 32 70 ul 201 077 HLA DRB1 07 09 R 40 Y 80 1 0 ul 12 30 ul 201 073 HLA DRB1 08 12 R 70 Y 140 1 6 ul 24 50 ul 201 074 HLA DRB1 11 R 95 Y 190 2 2 ul 30 70 ul 201 075 HLA DRB1 13 R 70 Y 140 1 6 ul 24 50 ul 201 076 HLA DRB1 14 R 75 Y 145 1 7 ul 27 55 ul 201 078 HLA DRB1 15 R 70 Y 140 1 6 ul 24 50 ul 201 079 HLA DRB1 16 R 40 Y 80 1 0 ul 30 ul 201 080 HLA DRB1 15 16 R 46 Y 94 1 1 ul 16 34 ul 7 10 Number and Positions of Primer Mixes of PROTRANS Domino System REF Protrans Number Position Position Domino System Primer Mixe 1 Primer Mix NC 201 071 HLA DRB1 01 13 1 13 201 072 HLA DRB1 03 20 1 20 201 070 HLA DRB1 04 32 1 32 201 077 HLA DRB1 07 09 12 1 12 201 073 HLA DRB1 08 12 24 1 24 201 074 HLA DRB1 11 30 1 30 201 075 HLA DRB1 13 24 1 24 201 076 HLA DRB1 14 27 1 27 201 078 HLA DRB1 15 24 1 24 201 079 HLA DRB1 16 12 1 201 080 HLA DRB1 15 16 16 1 16 User Manual PROTRANS HLA SSP Cyclerplate and Domino System Version 2 4 Rev 2009 10 20 page 10 of 16 8 Post PCR After thermal cycling remove the PCR Cyclerplate or Domino Strips and proceed to gel electrophoresis If not performing electrophoresis immediately store the Cyclerplate at 4 C for up to two days or at 20 C for longer 8
24. re PCR area All work carried out before PCR DNA isolation and storage preparations for the PCR production and storage of reagents and solutions for DNA extraction and PCR When working in the pre PCR area pipettes with aerosol protection should be used filtered tips It is recommended to use a negative control with every amplification to detect contaminations 6 3 Post PCR area Thermocycler gel electrophoresis evaluation storage of amplified DNA Equipment and consumables from the post PCR area must not be taken into the pre PCR area 7 Instruction for PROTRANS SSP DNA Typing Kits 7 1 Preparation of the PCR 7 1 1 Out of the refrigerator collect the desired PCR Cyclerplates or PCR Domino Strips Out of the freezer 20 C collect the DNA Taq Polymerase and T2 the corresponding Protrans Buffers See Tables 7 5 and 7 8 713 Place the PCR Cyclerplates or the PCR Domino Strips and the DNA Taq Polymerase into the cooled a 20 C PROTRANS PCR Workstation or into a 96 well PCR rack 714 With the Protrans Cutter scissors it is possible to cut the PCR Cyclerplates into single tests 7 1 5 Thaw the Protrans Buffers D or R and Buffer Y 7 2 Preparation of Master Mix for the Protrans PCR SSP System For each DNA sample to be tested pipette the appropriate volume of Buffer D or R and Buffer Y 121 and DNA Taq Polymerase into a 1 5ml reaction tube See Tables 7 6 and Table 7 9 7 2 2
25. resis Equipment REF 210 000 Incorrect camera setting Increase exposure time or aperture setting User Manual PROTRANS HLA SSP Cyclerplate and Domino System Rev 2009 10 20 15 of 16 Version 2 4 page Problem Possible Cause Solution Excessive amount of Ethidium bromide Use 5ul ethidium bromide 10mg ml for each Gel picture too bright used 100ml gel solution Incorrect camera setting Increase exposure time or aperture setting 11 Literatur References R f rences Bibliographiques Bibliografia 1 10 U Forssmann J Mytilineos S Scherer and G Opelz A method for HLA DQA Typing by the PCR SSP Technique Transplant Int 1994 7 515 518 J Mytilineos M Lempert D Middleton F Williams C Cullen S Scherer and G Opelz HLA Class DNA Typing of 215 HLA A B DR zero mismatched Kidney transplants Tissue Antigens 1997 50 355 358 J Mytilineos U Christ M Lempert and G Opelz Comparison of typing results by serology and PCR with sequence specific primer for HLA Cw in 650 individuals Tissue Antigens 1997 50 395 400 J Mytilineos M Lempert S Scherer V Schwarz and G Opelz Comparison of serological and DNA PCR SSP typing results for HLA A and HLA B in 421 black individuals Human Immunology 1998 59 512 517 J G Bodmer S G E Marsh E D Albert et al Nomenclature for factors of the HLA system 1996 Human Immunology V 53 98 129 March
26. rogram DNA concentration used was too high Repeat test with correct DNA concentration Wrong Taq Polymerase used Repeat test with validated DNA Taq Polymerase see 5 2 2 No visible control bands Inefficient thermocycler e g block defect Control of thermocycler by Protrans Cycler Check REF 200 100 Repeat test with validated Thermocycler see 5 2 3 The described program applies to the recom mended thermal cyclers Other thermal cycler as recommended have to be user validated User Manual PROTRANS HLA SSP Cyclerplate and Domino System Version 2 4 Rev 2009 10 20 page 14 of 16 Problem Possible Cause Solution No visible control bands Contact of the PCR plate to the block of the thermocycler is not correct The provided rack from PE sample holder must not be used in combination with the 96 well PCR Cyclerplates Incorrect Thermocycler program used Repeat test with correct amplification program No visible control bands continued Wrong Taq Polymerase used Repeat test with validated DNA Taq Polymerase see 5 2 2 In case of a homozygous typing result Repeat test DNA not evenly re suspended in original diluent or in Mastermix Vortex DNA sample and Mastermix thoroughly before PCR set up and repeat test If two alleles can be positively identified no action need to be taken In all other cases it is recommended to r
27. s or Protrans Coverplates from the Cyclerplates or Domino strips and pipette all PCR products with a 8 channel pipette into the slots of the gel Make sure that the order in which you load the samples is standardized The PROTRANS PCR Primer Mixes are containing Loading Buffer glycerol and cresol red don t use an additional loading buffer Caution Sudden movement of the Cyclerplate or Domino Strip can disperse amplified product contaminating the laboratory and may require repetition of the test To measure the size of the PCR Products a molecular weight marker standard 50 1000 bp ladder can be included in each row Run the PROTRANS Electrophoresis for about 20 min at 150 V the PROTRANS Gel Check for about 5 min at 200 V 8 3 Result Interpretation and result documentation Place the gel on a UV Transilluminator or on the PROTRANS UV Transilluminator suitable face protection against UV radiation should be worn For interpretation and documentation of the results place the PROTRANS Number Plate on the Gel and take a Polaroid picture 8 4 Result Interpretation Each of the PROTRANS primer mixes contains a non allelic amplification control primer pair which amplifies either a part 89 bp of the B globin gene HLA class or a part 440 bp of the C reactive protein gene HLA class Il The concentration of these primer pairs is lower than the allele specific primer pairs and their purpose is to provide an internal control of successful P
28. shorter control band 5 Some lanes have two or more different sizes of PCR products These wells have multiplexed primer pairs which give rise to different amplicons depending upon the allele present Refer to the locus specific tables in the LOT specific kit inserts 6 False negative reactions can be caused by e inefficient amplification e poor quality of DNA e uneven placement of the PCR plate in the block of the thermocycler e temperature variations across the wells of the thermal cycler itself e inadequate cycler calibration 7 Itis possible in rare instances that the false negatives are due to a new or yet uncharacterized allele In such cases it is recommended to repeat the test using another technique SBT 8 The test must be repeated if e absent control bands with no specific amplification are indicating failed reactions e there is an apparent homozygous result or the missed reaction could change an allele assignment e the reaction pattern gives no clear result e the reaction pattern shows the prescence of 3 alleles e the negative control is not negative 9 If alleles can be determined in the presence of a failed PCR reaction and that failed reaction does not change the allele assignment the test does not need to be repeated 10 For detailled informations refer to the HLA locus and LOT specific kit inserts User Manual PROTRANS HLA SSP Cyclerplate and Domino System Version 2 4 Rev 2009 10 20 page 12 of 16 9 Limitation
29. technology Symbols 2 3 3 3 1 Introduction 3 1 1 Intended Use 3 1 2 Summary 3 1 3 Principle of the Tests 3 2 Reagents 4 2 1 Contents of the Protrans SSP Kits 4 2 2 Warning and Precautions 5 2 3 Storage and shelf life 5 3 Instrument requirements 6 3 1 Programming the thermocycler 6 4 Specimen collection and preparation 6 4 1 DNA Isolation 6 4 2 Non acceptable specimens 6 5 Materials 7 5 1 Materials provided 7 5 2 Additional materials reagents and equipment required but not supplied 7 6 PCR Polymerase Chain Reaction 8 6 1 Precautionary measures 8 6 2 Pre PCR area 8 6 3 Post PCR area 8 7 Instruction for PROTRANS PCR SSP DNA Typing Kits 8 7 1 Preparation of the PCR 8 7 2 Preparation of the Master Mix for the PROTRANS PCR SSP System 8 7 3 Dispensing the Master Mix in the PROTRANS Cyclerplates or Domino Strips 8 7 4 Capping the PROTRANS Cyclerplates or Domino Strips 8 7 5 Table PROTRANS Cyclerplate System and PROTRANS Buffers 9 7 6 Table Volume of PROTRANS Buffers and Taq Polymerase for Mastermix 9 7 7 Table Number and Positions of Primer Mixes of PROTRANS Cyclerplate System 9 7 8 Table PROTRANS Domino System and PROTRANS Buffers 10 7 9 Table Volume of PROTRANS Buffers and Taq Polymerase for Mastermix 10 7 10 Table Number and Positions of Primer Mixes of PROTRANS Domino System 10 8 Post
30. x Buffer D and R Aliquot the Mastermix and freeze aliquots For the PROTRANS Cyclerplate System make aliquots of 280ul and freeze them For the PROTRANS Domino System make aliquots of 23 46 69 and 92ul and freeze them Dry PCR tubes after amplification Wells not sealed properly Repeat test and seal wells properly No visible bands in gel No Ethidium bromide in the gel Re stain gel Inefficient Thermocycler e g block defect Control of Thermocycler by Protrans Cycler Check REF 200 100 Repeat test with validated Thermocycler see 5 2 3 The described program applies to the recom mended thermal cyclers Other thermal cycler as recommended have to be user validated Incorrect Thermocycler program used Repeat test with correct amplification program Wrong Taq Polymerase used Repeat test with validated DNA Taq Polymerase see 5 2 2 DNA concentration used was too high low Repeat test with correct DNA concentration Degraded DNA smears in gel lanes Repeat test with a new re extracted DNA sample PCR inhibitors in the genomic DNA or impure DNA see 4 2 Repeat test with a new re extracted DNA sample out of EDTA Citrate blood Lack of DNA Taq Polymerase activity Repeat test with a known reference DNA to verify activity of Taq Polymerase Weak specific bands No control bands in the gel Inefficient Thermocycler e g block defect C
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