F A S T Y P E HLA -DNA SSP TYPING SYSTEM. User s Guide. For Class I HLA-ABC & Class II DR, DQ Low, Intermediate and High Resolution BIO SYNTHESIS

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1 F A S T Y P E HLA -DNA SSP TYPING SYSTEM User s Guide For Class I HLA-ABC & Class II DR, DQ Low, Intermediate and High Resolution BIO SYNTHESIS INCORPORATED Copyright Bio-Synthesis, Inc., All Rights Reserved

2 FASTYPE TM HLA-DNA SSP TYPING SYSTEM PROTOCOL. PRIMARY FEATURES OF THE FASTYPE TM HLA-DNA SSP TYPING SYSTEM:.. SIMPLE HLA DNA TYPING All primers, dntps, and buffers are pre-aliquot and lyophilized in high-quality PCR thermal cycler plates... FAST PCR REACTION SETUP Fastype TM HLA-DNA SSP Typing System Kits reduces the preparation time to less then 0 minutes... ENHANCED STABILITY Lyophilized primers, dntps and PCR buffer are more stable than in liquid form under ambient condition... EASY CONTAMINATION DETECTION Built-in contamination control primer pair(s) are designed to detect contaminations of genomic DNA and to detect carry-over PCR end products..5. UNIVERSAL PCR PROTOCOL The PCR Protocol for all Fastype TM HLA-DNA SSP Typing System is kept the same regardless of the kit type..6 UNION INTERNAL CONTROL BAND SIZE The internal control band is kept the same at 0bp for all Fastype TM HLA-DNA SSP Typing Kits to reduce the confusion of positive band versus internal control band.. PACKAGE CONTENTS, SHIPPING AND STORAGE CONDITIONS SHIPPING CONDITION: Ambient conditions/ room temperature. PACKAGE CONTENTS STORAGE SHELF LIFE FASTYPE TM KIT REAGENT E -0 O C -0 O C year or as specified on the package label year 0X DYE - O C year -WELL STRIP CAPS AMBIENT CONDITIONS. TYPING PROTOCOL. COMPONENTS OF FASTYPE TM HLA-DNA SSP TYPING SYSTEM KIT: a. Allele-specific or group-specific primer pair(s) b. Internal control primer pair specific for human GPDH gene c. dntps d. PCR buffer. MATERIALS (See Appendix B for required material and instrument). SETTING UP PCR (See Figure. for PCR parameters) --

3 FASTYPE TM HLA-DNA SSP TYPING SYSTEM PROTOCOL (BELOW STEPS ARE TO BE PERFORMED IN PRE-PCRAMPLIFICATION ROOM).. PREPARING REAGENT... Obtain a.5 ml- microcentrifuge tube.... Combine equivalent volumes of Reagent E and Taq Polymerase (5u/ul) into an : (v/v) mixture.... Incorporate the mixture in room temperature for 5 minutes. Store the remaining at -0 o C..5. PREPARING DNA MASTER MIX.5.. Remove Fastype TM SSP kits from -0 o C. Bring to room temperature. Secure on a 6-well microtiter plate holder. Note: You can mix and match different Fastype TM Typing Kits to maximize your typing needs..5.. Bring the Cresol red Dye (0X) to room temperature. Vortex thoroughly before using. Note: For short term storage, please kept at o C..5.. Obtain a.5ml microcentrifuge tube. Prepare Master Mix (See Appendix A for Master Mix formulation).5.5. Vortex the master mix thoroughly, spin down if necessary. Pipette 0ul into each well (or tubes). Important: Upon adding the master mix, the contents inside should turn into a bright pink color solution. If the solution turns yellow, your sample may be too acidic and may effect PCR amplification..6. PREPARING FOR CONTAMINATION CONTROL DETECTORS.. SEALING.6.. Prepare the following contents in built-in contamination control well (or tubes): Sterile ddi H O l 0X dye solution l Taq/Reagent E 0. l The final volume should add up to 0 l.... Gently tap the wall of the PCR tube to settle master mix to the bottom of the wells (or tubes).... Add one drop (~5 l) of mineral oil to each tube (optional).... Seal the reaction tubes with the caps provided or with a PCR mat... SECURE ON A THERMAL CYCLER AND BEGIN PCR CYCLE. FIGURE. PCR PARAMETER Thermal Cycler Protocol Program the thermal cycler with the following parameters: Tm Time Cycle No. Linked to Program 6 o C min Program Program Segment o C 0 sec Program Segment 65 o C 60 sec 0 Program Segment o C 0 sec Program Segment 6 o C 50 sec 0 Segment 7 o C 0 sec Program 7 o C 5 min --

4 FASTYPE TM HLA-DNA SSP TYPING SYSTEM PROTOCOL. AGAROSE GEL ELECTROPHORESIS THE AMPLIFIED BANDS CAN BE VISUALIZED BY SUBMARINE AGAROSE GEL ELECTROPHORESIS... PREPARING % (W/V) AGAROSE GEL... Dissolve.0 grams of electrophoresis grade agarose powder in 00 ml of 0.5X TBE buffer. (See Appendix B. for buffer preparation.)... Melt the agarose powder completely by boiling in a microwave oven or on a heating apparatus. Stir constantly. If evaporation occurs, replenish with ddi H O.. CASTING GEL... Cool the heated agarose gel to ~60 o C... Add at least ul of ethidium bromide (0 mg/ml ) to the heated agarose. Stir until the it is thoroughly incorporated.... On a balanced surface, set up a gel plate with combs containing a minimum of wells per row.... Cast a 5mm thick gel on the plate...5. Allow the gel to settle. (BELOW STEPS ARE TO BE PERFORMED IN POST-PCR AMPLIFICATION ROOM). GEL ELECTROPHORESIS... Submerge the gel in 0.5X TBE buffer in a gel box.... Gently remove the caps to avoid splashing of PCR products.... Start pipette from the bottom-left of the kit and proceed to the right and upward. (Refer to Fastype Kit Configuration for details)... Load a minimum of l into each well on the gel. Note: The PCR products are stained with cresol red. There is no need to load additional loading buffer or dye...5. (Optional) Reserve the first well on each row for molecular weight standards, in increments of 00bp from 50 to 000bp...6. Connect the electric leads and turn on the power supply (5V AC ). Electrophoresis for ~ twenty (0) minutes, or until two thirds (/) of the lane.... Turn off the power supply, and remove the gel from the gel box.. VISUALIZING RESULTS... Transfer the gel onto a UV transilluminator.... Document the result by photography.... Use the corresponding typing sheet and table to interpret results. 5. INTERPRETATION OF THE RESULT Attention: Due to continuous updating, the typing sheet and specificity table may vary from lot to lot. Upon receiving, please confirm the lot number on the package label matches the typing sheet and specificity table provided. Should you have any questions, please contact our Technical Support at Refer to Typing Sheet and Specificity Table for product sizes and primer information. 5.. All Internal control band has a size of 0 bp. The intensity of the internal control PCR product may vary due to the presence of positive amplifications. 5.. If the PCR products have bands other than the predicted size, disregard the band for it may be none specific or primerdimer artifacts. 5.. The presence of a 5bp band in contamination control lane implies a contamination of Post-PCR Product. The presence of a 0bp band alone or accompanied by a 5bp band implies a contamination of genomic DNA. --

5 TROUBLE SHOOTING AND TECHNICAL GUIDE 6. TROUBLE SHOOTING GUIDE PROBLEMS. The control and specific bands are weak.. Heavy smear of bands or dim specific bands. Missing internal control bands in one or several lanes. False negative of a specific band while the internal control appears normal. 5. More than two specific alleles are detected 6. Ambiguous results 7. Blurred bands 7. TECHNICAL GUIDE POSSIBLE CAUSE a. Concentration of DNA sample is too low. b. Degradation of sample DNA. c. Inhibitors for Taq polymerase present in the DNA sample. Template DNA is in excess. a. The electrophoresis buffer might become too hot due to high voltage. b. Wrong buffer composition. c. Old buffer SUGGESTIONS a. Check DNA quality and concentration (see Technical Guide). b. Re-purify the sample DNA and dissolve it in ddiho. Check DNA concentration and adjust accordingly. Repeat typing with another good quality DNA sample. Repeat typing with a control DNA sample which has the same specific allele. a. Separate the pre-pcr room from the post-pcr room. b. Kept a different lab coat between rooms. c. Clean the working area with 0% bleach. d. Change protective gloves frequently, especially transfer from post- PCR room to pre-pcr room. Report to Technical Support a. Lower the voltage during gel electrophoresis. b. Use the recommended 0.5 x TBE buffer. 7-. DNA Quality For optimal results with the Fastype TM DNA typing system, the quality of sample DNA is critical. Good quality DNA means the 60/0 ratio of OD is higher than.6 and the major portion of DNA should run higher than. kb on an agarose gel. Heavy degradation of genomic DNA or a 60/0 ratio lower than.5 requires DNA re-extraction. For best result, dilute sample DNA in ddiho. 7-. DNA Quantity The quantity of sample DNA should be 5-50 ng per reaction. Excess of sample DNA (00 ng or more) can cause decline of specific PCR products. An intense smear of high molecular DNA is an indication of excess of sample DNA. RNA contamination can cause higher readings of DNA concentration with a spectrophotometer. If PCR reactions fail, it is always recommended to check the quantity and quality of sample DNA by running sample DNA in % agarose and compare it with a series of standard Lambda DNA marker. 7-. Taq polymerase Fastype TM kits have been intensively tested with the AmpliTaq DNA polymerase from Perkin-Elmer. We recommend that users use AmpliTaq DNA polymerase as the first choice. Do not use Tth DNA polymerase. 7-. Amplification Procedure At the end of PCR, examine the degree of evaporation and condensation of PCR reaction mixture. If there is more than 0% volume loss, you should overlay the PCR reaction mixture with mineral oil. It is also a good practice to maintain QC records on the heating lid. If the temperature of the heating lid is not high enough, it will cause condensation problems on the lid Thermal cycler Perkin-Elmer 600 and 700 thermal cyclers are recommended for Fastype HLA Class I and Class II typings. IF PROBLEMS STILL PERSIST, CALL THE TECHNICAL SUPPORT AT Contamination with previously amplified PCR products or with other DNA samples during the DNA extraction or PCR preparation. --

6 FASTYPE KIT CONFIGURATION Kit Type Kit Type FASTYPE TM KIT CONFIGURATION HLA-A (cat FA) Color Code: Blue No. of wells: DRB*0 (cat. 0-0-F) Color Code: Purple No. of wells: HLA-B (cat FB) Color Code: White No. of wells: 7 HLA-C (cat FC) Color Code: Yellow No. of wells: DR Low (cat FR) No. of wells: DQ Low ( cat FQ) No. of wells: DQ High (000) (cat FQH) No. of wells: DQ Intermediate (Previously DQ High) (cat FQI) No. of wells: DQA (cat FQA) No. of wells: DRB* 0 (cat. 0-0-F) Color Code: White No. of wells: DRB*0 (cat. 0-0-F) Color Code: Blue No. of wells: 6 DRB* 07 ( tests per strip) (cat F) DRB*0 (cat. 0-0-F) Color Code: Yellow No. of wells:6 DRB* (cat. 0--F) No. of wells: 6 DRB* (cat. 0--F) No. of wells: DRB* (cat. 0--F) Color Code: White No. of wells: 6 DRB* (cat. 0--F) Color Code: Blue No. of wells: 6 DRB*5 (cat. 0-5-F) Color Code: Purple No. of wells: DRB*6 (cat. 0-6-F) No. of wells: DRB (cat. 0-B-F) Color Code: Yellow No. of wells: DRB (cat. 0-B-F) No.of wells: DRB5 (cat. 0-B5-F) Color Code: White No.of wells: cc NOTE:. refers to built-in contamination control.. The COLOR CODE refers to the plate color, the ring color if it is a -well strip, or unless specified otherwise. -5-

7 MASTER MIX GUIDE Appendix A S a m p le DN A d d i H O ( ~ 5 0 n g /u l) n C r e s o l Re d Dye (0 X ) T a q + R e a g e n t E m i x t u r e (5u n i t s / u l ) T o t a l V o lu m e (u l ) (u l ) (u l ) (u l ) (u l ) Note: n is the number of reactions. To compensate for pipette variations, each master mix above ( where n > 6) is adjusted with extra reaction to (n+). -6-

8 Appendix B TABLE. INSTRUMENTS AND MATERIALS TABLE. ELECTROPHORECTIC SOLUTIONS PCR Amplification Thermal Cycler Taq Polymerase Sterile Water Gel Electrophoresis Gel box Agarose Ethidium Bromide 0.5X TBE Buffer Microwave oven / Heating Equipment Stir Plate Power Supply (5V AC 50-60Hz) UV Trans-illuminator Polaroid camera/film or Video camera/printer Recommended Suppliers Perkin-Elmer GeneAmp PC system 600 or 700 AmpliTaq DNA Polymerase Perkin-Elmer N Bio-Typer TM Electrophoresis System / Life Technologies (electrophoresis grade) Sigma E-50 0X TBE Buffer L ph.0 Tris-base 0g Boric Acid 55g EDTA. g (Sodium Salt) Dissolve all ingredients in ddiho and adjust the volume to L. Filter the solution to remove particles. Store in bottles at room temperature (5 o C). 0.5X TBE Buffer Dilute 500ml 0X TBE buffer to 0 L ddiho Store in ambient condition. Ethidium Bromide Solution 00 ml Ethidium Bromide g Dissolve EtBr in 00ml ddiho Stir for several hours until the dye has dissolved completely. Store in an amber bottle or wrap with aluminum foil to avoid light. Store in ambient condition, up to months WARNING! Ethidium bromide is a powerful mutagen! Handle with care. PCR LICENSE The use of polymerase chain reaction (PCR) for in-vitro diagnostic procedure requires a license. The PCR is covered by United States Patents,6,5 and,6,0 and corresponding patent rights in other territories, owned by F. Hoffmann-La Roche Inc. License can be obtained from: Director of Licensing Roche Molecylar Systems 5 Atlantic Avenue Alameda, CA 50 USA -7-

9 REFERENCES 0. REFERENCES. Bidwell, J.H. Application of the Polymerase Chain Reaction to HLA Class II Typing, Vox Sang 6: -. (). Bunce, M., et. al. Phototyping: comprehensive DNA typing for HLA-A, B,C DRB, DRB, DRB, DRB5 & DQBby PCR with primer mixes utilizing sequence-specific primers (PCR-SSP). Tissue Antigens 6: (5). Chen, R., et.al. High Resolution Typing of HLA-DR by PCR amplification with sequence -specific primers (PCR-SSP). Human Immunology nd Annual ASHI Metting Abstracts 7. #: (6). Innis, M.A., et. al. PCR Protocols: A guide to methods and applications. Academic Press, San Diego, CA Olerup, O., et. al. HLA-DR typing by PCR amplification with sequence-specific primers (PCR-SSP) in hours: An alternative to serological DR typing in clinical practice including donor-recipient matching in cadaveric transplantation. Tissue Antigens : 5-5. () 6. Olerup, O., et. al. HLA-DQB and DQA typing by PCR amplification with sequence-specific primers (PCR-SSP) in hours. Tissue Antigens : -. () 7. Robinson J, Malik A, Parham P, Bodmer JG, Marsh SGE: IMGT/HLA database - a sequence database for the human major histocompatibility complex. Tissue Antigens (000) Saiki, R.K., et. al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase, Science : 7-. (). Sambrook, J. et. al. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,. 0. Zetterquist, H. et. al. Identification of the HLA DRB*0, -DRB*07 and -DRB*0 alleles by PCR amplification with sequence-specific primers (PCR-SSP) in hours. Human Immunology : 6-7. () LIMITED WARRANTY &TERMS Fastype TM system products have a one year manufacturer warranty. This warranty is valid only to the original purchaser for one year from the date of purchase. At Bio-Synthesis discretion, Bio-Synthesis obligation to the original purchaser is limited either the replacement of the product without extra charge, or a total reimbursement. The purchaser is responsible for the retuning of any defective items to Bio-Synthesis Incorporated. All shipment must be such that no damage is incurred. Claims for shipping damage must be submitted to the transit carrier. This warranty becomes null and void when any product(s) have been modified, repackaged or resold without Bio-Synthesis consent, or when any product(s) have been subjected to misuse or mishandling. Bio-Synthesis Incorporated will replace or refund to the original purchaser if the purchaser is dissatisfied with the overall performance of Fastype TM system within the first thirty (0) days of purchase. If the purchaser has any other concern, please contact our service support at

This Product Description is only valid for Lot No. 36V.

This Product Description is only valid for Lot No. 36V. HLA Wipe Test Negative Control Product Insert Page 1 of 8 Olerup SSP HLA Wipe Test Negative Control Product number: 102.101-01 including Taq polymerase Product number: 102.101-01u without Taq polymerase