LM DNA Kit Product Insert
Contents
1. Veriti 96 Well Thermal Cycler Table 3 Thermal Cycler Conditions for Amplification Step Temperature and Incubation Time of Cycles 1 95 C for 3 min 1 95 C for 15 sec 2 60 C for 30 sec 12 72 C for 30 sec 95 C for 10 sec 3 63 C for 30 sec 28 72 C for 30 sec 4 72 C for 2 min 1 5 4 C forever 1 Note To be sure of sample amplification refer to Product Gel Electrophoresis Appendix A C H UN oi ybridization Be sure hybridization buffer components of the LIFECODES probe mix are solubilized and that the beads are thoroughly suspended Turn on the Luminex Instrument and XY Platform to allow for 30 minute warm up Warm probe mix in a 55 to 60 C heat block for at least 5 10 minutes to thoroughly solubilize components in probe mixture Sonicate briefly 15 sec then vortex probe mix for about 15 seconds to thoroughly suspend the beads Combine 15 uL of the appropriate probe mix with 5 uL of locus specific PCR product into each well of a thermal cycler 96 well plate Costar No 6509 When aliquoting probe mix to more than 10 wells gently vortex probe mix after each set of ten Seal plate with polyethylene tape Costar No 6524 Place silicone compression mat on top of plate prior to hybridization Hybridize samples under the following incubation conditions Table 4 Thermal Cycler Conditions For Hybridization 97 C for 2 minutes 47 C f2. Diagnostics by Promega Corp It is licensed to Promega under U S Patent Nos 5 338 671 and 5 587 287 and their corresponding foreign patents The purchase of this product includes a limited non transferable license under U S patent 5 981 180 or its foreign counterparts owned by Luminex Corporation MANUFACTURER INFORMATION Manufacturer Immucor Transplant Diagnostics Inc 550 West Avenue Stamford CT 06902 Phone 203 328 9500 888 329 0255 Fax 203 328 9599 European Technical Service Phone 32 3 385 4791 This document last revised and issued TRADEMARKS USED AB Gene AB Gene House Luminex Luminex Corporation Costar Corning Incorporated Gene Amp Roche Molecular System Microseal Bio Rad Laboratories Inc Veriti Applied Biosystem IDNA Agarose Lonza Group Ltd LIFECODES Immucor Inc GelStar Lonza Group Ltd Page 8 of 9 LC1436RUO 4 05 15 APPENDIX A Gel Electrophoresis The PCR reactions performed in the LIFECODES HLA SSO Typing Kits are designed to produce both double and single stranded products which are the predominant products that hybridize to the SSOs For quality assurance or to trouble shoot an experiment it might be necessary to perform gel electrophoresis to examine the PCR reaction for the presence of amplified DNA Materials Required as listed or equivalent Electrophoresis Grade Agarose Lonza Group Ltd IDNA e GelStar Nucleic Acid Gel Stain Lonza Group Ltd No 50535 Agarose
3. No 50170 UV Transilluminator ChromatoVUE UVP Inc Model TM36 Electrophoresis apparatus power supply Photographic imaging system e 1X Gel Buffer 40xTAE Promega No V4281 The relative migration of the single stranded product is dependent upon the gel concentration and buffer system employed Approximate migrations for each amplification are listed below for samples run in a 2 Agarose gel in 1X TAE buffer Electrophoresis Conditions Note Not applicable for HLA DQA1 B1 Part 628930R and HLA DPA1 B1 Part 628936R as individual bands cannot be distinguished in combination multiplex 1 Remove GelStar Nucleic Acid Stain Lonza Group Ltd No 50535 from freezer to thaw Keep in dark 2 The gel used for this procedure must be 2 i e for a 200ml gel bed use 4 grams of agarose to 200mL 1X TAE Dilute from 40X TAE Add 10uL GelStar Nucleic Acid Stain to the molten agarose When pouring the gel be sure to leave ample room for DNA to run a significant distance 1 to 2 inches USE CAUTION GelStar is a potential Carcinogen NOTE It is possible to run gels with 20uL of 10mg mL Ethidium Bromide in place of GelStar Nucleic Acid Stain Product band intensity will be less in gels containing Ethidium Bromide than in gels containing GelStar USE CAUTION Ethidium Bromide is a known Carcinogen 3 Keep gel in dark and allow to solidify 4 Load a mixture of 2 5uL of each PCR product and 2 5uL 2X loading buffer with visible dye p
4. affected by the type of biological material method of purification amount and integrity of genomic DNA Therefore the signal obtained for the Consensus probe s can serve as an indicator of the success of the amplification and hybridization procedures Also the signal obtained with the Consensus probe can be used to normalize the signal of the allele specific probes and correct for variations in the amount of amplified product in the hybridization reaction The analysis of the results generated from the SSO typing can be used to determine the presence or absence of particular DNA sequences in amplified DNA and to identify the possible alleles in the sample For the LIFECODES HLA SSO Typing procedure probes are attached to Luminex Microspheres designed for use with the Luminex Instrument Up to 100 different populations of Luminex Microspheres can be mixed together and analyzed by the Luminex Instrument because each population of microspheres can be distinguished by its unique fluorescence signature or color A different SSO probe can be attached to each color microsphere Therefore a mixture of several probes can be distinguished from each other by virtue of their association with particular color microspheres The Luminex Instrument is also able to quantify the relative amounts of labeled PCR product hybridizing to each Luminex Microsphere Therefore the relative signal obtained with the SSO probes in the LIFECODES assay as with other SSOP methods can b
5. far ase for Use Page 1 of 9 LC1436RUO 4 05 15 KITS REAGENTS BY CATALOG NUMBER LIFECODES HLA DQA1 B1 SSO Typing Kit Catalog 628930R LC DQAB LIFECODES HLA DQA1 B1 Typing Kit for use with Luminex Product 200210 50R CDGAB LIFECODES HLA DQAT B1 Master Mix 870 uL Vy 50 LIFECODES HLA DQA1 B1 Probe Mix 2 to 8 C CEON ps Dilution Solution 628515 19 7 mL 18 to 30 C TAQ LIFECODES Taq Polymerase 628076 25 uL 10 C to 30 C a ee LCT DPAB LIFECODES HLA DPA1 B1 SSO Typing Kit Catalog 628936R LC DPAB LIFECODES HLA DPA1 B1 Typing Kit for use with Luminex Product 200110 50R MX DPAB LIFECODES HLA DPA1 B1Master Mix 200100 870 pL y 50 l 2 to 8C Sufficient for 50 BM DPAB LIFECODES HLA DPA1 B1 Probe Mix 200101 810 uL Protect From Light samples Dilution Solution 628515 19 7 mL 18 to 30 C TAQ LIFECODES Taq Polymerase 628076 25 uL 10 C to 30 C ihe se LCT DQA LIFECODES HLA DQA1 SSO Typing Kit Catalog 628933 LM DQA LIFECODES HLA DQA Typing Kit for use with Luminex Product 628061R Reagent Product Number Fill volume y 20 MX DQA LIFECODES HLA DQA Master Mix 628062 420 uL Sufficient for 20 samples 2 to 8 C BM DQ LIFECODES HLA DQA Probe Mix 628063 360uL protect From Light Dilution Solution 628155 18 to 30 C TAQ LIFECODES Tag Polymerase 628076 25 uL 10 C to 30 C Do ose LCT DPB LIFECODES HLA DPB1 SSO Typing Kit Catalog 628937 LM DPB LIFECODES HLA DPB Typ
6. IMMUCOR LIFECODES Immucor Transplant Diagnostics Inc 550 West Avenue Stamford CT 06902 USA Tel 203 328 9500 or 888 329 0255 Fax 203 328 9599 WWW IMMUCOR COM Product Documentation available at www lmmucor com PRODUCT INSERT LIFECODES HLA SSO TYPING KITS For RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC PROCEDURES TABLE OF CONTENTS Definition of Symbols Directions for Use Reagents by Catalog Number A Purify Genomic DNA B Amplification Summary and Explanation C Hybridization Principles of the Procedure D Analyze with Luminex Instrument Results Identification Quality Control Warnings and Cautions Limitation of the Procedure Storage Instructions Troubleshooting Purification or Treatment for Use Expected Values Instability Indications Reagents ON NN ODO A A DB Instrument Requirements References Specimen Collection and Preparation Limited License Information Procedure A Materials Provided Trademarks Used Appendix A B Materials Reagents and Equipment required Gel Electrophoresis but not provided Gel Interpretation C Additional Materials to be provided by the User DEFINITION OF SYMBOLS Product Labels and Supplemental Documents Catalog REF Temperature Batch Code LoT Number REF limitations Upper limit of temperature Sufficient for N Tests Do Not Freeze Use By Keep away Date from light Caution Consult See instructions Manufacturer Instructions
7. Luminex Instrument For best results assay the samples immediately using the Luminex Instrument Samples can be read up to 30 minutes after being diluted If not read immediately protect samples from light 1 Turn on the Luminex Instrument between 30 minutes and 4 hours before assaying the samples 2 Prior to analyzing the samples on the Luminex Instrument set up a Batch Run by which the samples will be analyzed a Select Create a New Batch from the File menu For example if analyzing for HLA DPB1 add Batch for HLA DPB1 The Batch Template is provided and is named in this case HLA DPB1 Please note that the template versions are lot number specific and correspond to the kit lot numbers Follow the stepwise instructions that appear on the screen for creating batches When naming the batch do not include commas in the name because information after a comma will be lost upon exportation of the data For further instructions on creating batches and multibatches refer to the Luminex User s Manual b Click the eject icon to eject the plate holder Place the 96 well thermal cycler plate containing the samples in the XYP heater block present on the plate holder Click the Retract icon The samples are now ready to be analyzed A prime step should be performed before starting the run d After the samples have been run through the instrument a sanitization step with 70 Isopropanol or 20 household bleach should be performed followed by two wash s
8. and tips 1 20uL 20 200uL 1000uL Spreadsheet analysis software Heat Block 70 Isopropanol or 20 Bleach Retainer tray Applied Biosystems 403081 for use with the 9700 thermal cycler only DIRECTIONS FOR USE NOTES Probe mixes and SA PE are light sensitive keep away from light and do not freeze Warm beads at 55 to 60 C for at least 5 10 minutes to thoroughly solubilize components in probe mixture Sonicate briefly 15 sec then vortex probe mix for about 15 seconds to thoroughly suspend the beads Take extreme caution in the aliquoting process using calibrated pipettes Failure to do so may result in reagent loss and sample failure All temperatures must be precisely maintained Fluctuations as little as 0 5 C can affect results At the hybridization stage samples should not remain in the diluted state at 56 C for more than 5 minutes see Results section It is recommended to assay the amplified samples as soon as possible If the samples cannot be run on the Luminex Instrument the same day the amplified product can be stored up to 3 days at 2 8 C prior to use For longer storage store at 20 C up to one week until ready to assay The amplified product can only be frozen and thawed once Repeated freezing and thawing will result in degradation of amplified samples and will yield poor results if assayed A Purify genomic DNA using method of choice final concentration should be 10 to 200 ng uL Adjust if necessa
9. ated within manufacture s prescribed parameters 1 Beads must be pre warmed and well suspended prior to use This ensures that the hybridization buffer components are in solution 2 47 C and 56 C incubations require a high degree of accuracy 0 5 C A thermal cycler should be employed Temperature should be verified within wells of the 96 well thermal cycler plate using a thermocouple e g Bio Rad Model VPT 0300 or equivalent The temperature within wells and among wells should not vary more than 0 5 C 3 Time at 56 C is critical and should not exceed a total of 13 minutes This includes the 8 minute incubation plus no more than 5 minutes to dilute all the samples with Dilution Solution SA PE mixture 4 Once diluted the samples are stable at room temperature 18 to 30 C for up to 2 hours protect from light Since a full 96 well plate can take up to 1 5 hrs To run through the Luminex Instrument the analysis should be started no more than 30 minutes after dilution to ensure that the last sample is analyzed within the 2 hr limit 5 Do not mix components from other kits and lots Due to the complex nature of HLA typing qualified personnel should review data interpretation and typing assignments TROUBLESHOOTING Low Bead Count Probe Mix not well suspended Prewarm sonicate and vortex probe mix and repeat assay Instrument not Out of Calibration Calibrate Instrument Refer to Luminex IS User s functioning properly ma
10. awing should be avoided since this can result in DNA degradation Page 3 of 9 LC1436RUO 4 05 15 PROCEDURE A Materials Provided See tables in Reagents by Catalog Number section for specific information Appropriate Master Mix MX Threshold Table s Probe Hit Chart s Appropriate Probe Mix BM LIFECODES Taq Polymerase LIFECODES Cat No 628076 Dilution Solution DS B Materials Required but Not Provided The following are recommended for use with the kit Luminex Sheath Fluid 1x LIFECODES Cat No 628005 e Thermowell Clear Polyethylene Tape Costar No 6524 Nuclease free water LIFECODES Cat No 757003 20mL LIFECODES Cat No 888635 PCR tubes and caps Corning Thermowell Tube Strips e R Phycoerythrin Conjugated Streptavidin SA PE 1mg mL Costar Cat No 6542 LIFECODES Cat No 888640 or LIFECODES Cat No 628511 Corning Thermowell PCR 96 well plates Cat No e Luminex Calibration Kits Luminex 100 200 Calibration Kit CLS6551 or Themoscientific AB Gene Superplate 96 well Luminex 100 200 Performance Verification Kit LIFECODES PCR plate Cat No AB 2100 Cat Nos 628018 and 628019 respectively Costar plate Costar Cat No 6509 LIFECODES Cat No 888630 C Additional Materials to be provided by the user Vortex Mixer Silicone compression Mat Axygen Scientific CM FLAT or equivalent Bath Sonicator Microcentrifuge Barrier filter tips Pipettors Multichannel pipettors
11. e used to assign the probes as having positive or negative reactivity with the amplified DNA sample see Results section This in turn provides the information needed to determine the HLA phenotype of the sample REAGENTS A Identification See tables in Reagents by Catalog Number section for complete listing of products and catalog numbers B Warnings or Cautions 1 For Research Use Only Not for use in diagnostic procedures 2 Separate pipettes should be designated for Pre PCR manipulations as well as for Post PCR manipulations 3 Biohazard All biological and blood samples should be treated as potentially infectious Use Universal Precautions when handling 4 Dilution Solution Probe mixes Taq Polymerase and R Phycoerythrin Conjugated Streptavidin contain hazardous compounds Avoid contact with skin and eyes and dispose of all materials after use according to local regulations See Material Safety Data Sheets for additional information C Storage Instructions 1 Refer to kit component packaging label for proper storage temperatures 2 Probe mixes and R Phycoerythrin Conjugated Streptavidin are light sensitive KEEP FROM LIGHT DO NOT FREEZE 3 Do not use components past their expiration date D Purification or Treatment Required for Use See Specimen Collection and Preparation E Instability Indications 1 If salts have precipitated out of solution during shipping or storage resolubilize completely prior to use by vortexing at room te
12. er sample per amplification Let gel run in the dark at approximately 160 volts for 45 minutes or until sample runs far enough to see separate bands for single and double stranded product bromophenol blue band or other visible marker migrates 1 to 2 inches from wells 5 Photograph using UV Transilluminator accompanied by a GelStar Yellow Photographic Filter Lonza Group Ltd No 50536 CAUTION Wear protective equipment when handling GelStar Nucleic Acid Stain or Ethidium Bromide and when photographing gel using UV Transilluminator HLA DPB HLA DQA 6 Gel analysis Double Strand s bp 280 240 Single Strand s bp 180 160 Gel Interpretation Amplification Non amplification Well Double Stranded DNA Bright Single Stranded DNA _ less bright Primer Band Page 9 of 9 LC1436RUO 4 05 15
13. from the sample values producing the background corrected data set Background Control values are found in the Threshold Table and are lot specific Background values are average MFI values for each bead to compensate for background noise due to bead variation 4 For each sample divide the background corrected data for each probe by the background corrected value for the corresponding consensus probe producing the normalized data set MFI Probe MFI Control blank for probe MFI Consensus MFI Control blank for consensus 5 For each probe record the normalized value on the Threshold Table Worksheet 6 Once all values have been assigned the probe hit pattern i e the combination of all positive and negative assignments for a given sample can be compared with the Probe Hit Table s provided Caution e There is a separate threshold table for each locus and each probe mix These threshold tables are Lot specific be certain that the Lot on the threshold tables matches the Lot in the typing kit Page 6 of 9 LC1436RUO 4 05 15 e Ifa normalized value for a particular probe falls above the maximum threshold for a negative assignment and below the minimum value for a positive assignment the sample should be considered as indeterminate for this probe The sample should be typed first assuming the value to be negative and then again assuming the value to be positive e See EXPECTED VALUES section for further information on th
14. ing Kit for use with Luminex Product 628910 R Mx DPB LIFECODES HLA DPB Master Mix 628903 420 uL Sufficient for 20 samples 2 to 8 C BM DPB BM DPB LIFECODES HLA DPB Probe Mix 628902 360 uL Protect From Light fs Dilution Solution 628155 9 9mL 9 9 9mL 180300 to 180300 TAQ LIFECODES Tag Polymerase 628076 25 uL 10 C to 30 C ae eel ne Probe Mixes are light sensitive keep exposure to light at a minimum CAUTION Do not use components past their expiration dates Page 2 of 9 LC1436RUO 4 05 15 SUMMARY AND EXPLANATION DNA based HLA typing using PCR amplified DNA is a common laboratory procedure PCR amplification of DNA is used as the means to enrich for a selected DNA region For HLA typing a subsequent assay is utilized to determine the properties of the amplified DNA Several types of assays such as SSP 1 direct SSOP 2 RFLP 3 and reverse SSOP dot blot technologies 4 have been used in HLA typing Like SSOP and reverse dot blot methods LIFECODES HLA SSO Typing kits utilize sequence specific oligonucleotides SSOs to identify which HLA alleles are present in a PCR amplified sample It is the set of SSOs employed not the methodologies that determines the ability to distinguish among the various alleles present in the PCR amplification Whereas reverse dot blot and SSOP methods employ enzyme labels and colorimetric substrates that require subsequent development the LIFECODES assay is a homogenous multiple
15. mperature 18 to 30 C 2 Donot use R Phycoerythrin Conjugated Streptavidin that has been frozen during shipment or storage INSTRUMENT REQUIREMENTS 1 Luminex Instrument and XY Platform Product Number 888300 888310 2 The following Thermal Cyclers have been validated 96 Well GeneAmp PCR System 9700 set to MAX mode Base Cat N8050200 Gold Block Cat 4314878 Veriti 96 Well Thermal Cycler set to 9700 MAX mode Cat 4375786 Refer to Table 2 for maximum ramp speeds SPECIMEN COLLECTION AND PREPARATION a Human DNA can be purified from Whole blood Buffy coats and Buccal swabs using a validated method that meets the criteria below b DNA extracted from blood preserved in EDTA and ACD Acid Citrate Dextrose have been tested and shown to yield expected performance in this assay DNA extracted from blood preserved in heparin cannot be used in this assay Other preservatives have not been tested c The isolated DNA should be in 10 mM TRIS pH 8 0 9 0 or in nuclease free water If a chelating agent such as EDTA is present the final concentration of the chelating agent should not exceed 0 5 mM d The presence of alcohol detergents or salts may adversely affect DNA amplification e Final DNA concentration should be 10 to 200 ng uL f Absorbance measurements of the DNA sample at 260 and 280nm should give a ratio of 1 65 to 2 0 g DNA can be used immediately after isolation or stored at 20 C for up to 1 year Repeated freeze th
16. nual Sample flow path blocked Remove and sonicate needle Perform backflush Call Immucor Transplant Diagnostics Inc if problem persists 888 329 0255 CON Threshold Sample failed to amplify Low DNA Check DNA concentration and purity Failure or amplified poorly Salts in Master Mix are out of Heat Master Mix at 37 C for 5 minutes vortex gently solution and spin down briefly Poor Tag Polymerase Use LIFECODES Taq Polymerase Catalog 628076 Amplification conditions not within specific parameters Run Thermal profile on Thermal cycler to verify parameters are within specified parameters Low Median Fluorescent Intensity Value MFI Warm dilution solution at 45 C for 5 minutes before use and vortex Store at room temperature Replace R Phycoerythrin Conjugated Streptavidin Multiple SSO Allele specific Amplification conditions not Run Thermal profile on Thermal cycler to verify failures or sample amplification within specific parameters parameters are within specified parameters fails to yield a HLA DNA sample contaminated typing result DNA parlially degraded Re isolate DNA from Blood sample Evaporation during hybridization step If not using an entire plate leave one row empty on each side of samples to be assayed to allow plate to be sealed tightly PCR amplification can be verified by gel electrophoresis See Appendix A Page 7 of 9 LC1436RUO 4 05 15 EXPECTED VALUES Values typically can be resolved as p
17. or 10 minutes 56 C for 8 minutes 562 C HOLD Ensure that the detection laser on the Luminex Instrument is turned on at least 30 minutes before the hybridization ends While the samples are hybridizing prepare a 200 1 Dilution Solution SA PE mixture Combine 170uL Dilution Solution DS and 0 85uL 1mg mL SA PE per sample Itis recommended to make enough Dilution Solution Mixture for n 1 samples to account for pipetting loss See Table 5 Keep Dilution Solution SA PE mixture in the dark at room temperature SA PE is light sensitive The Dilution Solution may be warmed at 45 C for 5 minutes and vortexed upon arrival to ensure all components are in solution Dilution solution must be at room temperature 18 to 30 C before making the mixture Prepare prior to use and discard any remaining portion Page 5 of 9 LC1436RUO 4 05 15 Table 5 Dilution Solution Preparation Volumes of Samples Dilution Solution DS SA PE ar a 170pL 0 85uL C o 850uL 4 25uL 1700uL 3400pL 8500uL 42 5uL Note DO NOT CANCEL HYBRIDIZATION PROGRAM BEFORE REMOVING THE TRAY FROM THE THERMAL CYCLER 8 At the 56 C hold while the tray is on the thermal cycler dilute each sample with 170uL of the prepared Dilution Solution SA PE mixture It is critical to dilute all samples within 5 minutes following the 8 minute 56 C HOLD step 9 Remove the sample tray from the thermal cycler and place in the Luminex Instrument D Analyze sample using the
18. ositive and negative In some rare instances the values may be indeterminate An indeterminate value represents a range in which neither positive nor negative values have been observed If a sample contains indeterminate values for a particular SSO probe the sample should be typed with the probe as a negative and again with the probe as a positive Three cases can arise 1 One of the choices i e Positive or Negative provides a match 2 Both choices provide matches e Sample can be re assayed or the alleles obtained from both typings can be reported 3 Neither choice produces a match e In this instance there are likely to be other probes that were incorrectly assigned This sample needs to be re assayed and possibly re amplified e f more than two probes are indeterminate the sample should be re assayed e If a probe hit pattern does not produce an HLA type the sample should be re amplified and re assayed It may also be necessary to re isolate DNA from the sample and reamplify and reassay e As noted in the Limitations of the Procedure section it is critical to precisely follow the protocol Any deviations can lead to sample typing failure REFERENCES 1 Olerup O et al 1992 Tissue Antigens 39 225 3 Maeda M et al 1989 Tissue Antigens 34 290 2 Saiki RK et al 1986 Nature 324 163 4 Bugawan TL et al 1990 Immunogenetics 32 231 LIMITED LICENSES Taq polymerase is manufactured for Immucor Transplant
19. reshold values QUALITY CONTROL It is recommended that one negative and positive control be run with each test such as a water blank and a previously typed sample respectively Consensus SSO probes listed on the Threshold Table hybridize to their respective locus specific alleles Values obtained with the Consensus SSOs from positive controls should exceed the threshold value for the SSO as set forth in the Threshold Table Worksheet Values obtained with the Consensus SSOs from water blanks should be below the threshold value for the SSO as set forth in the Threshold Table Worksheet The LIFECODES Probe Mix es contain one or more consensus SSO probes identified in the typing kit worksheets These consensus probes hybridize to all alleles and act as internal controls to verify amplification and to confirm that hybridizations occurred If the minimum value is not obtained for these SSOs the sample may not produce the correct typing and the sample test should be repeated The assay should be run as recommended in the package insert as well as performed with any other quality control procedures that are in accordance with local state federal and or accreditation agencies requirements LIMITATIONS OF THE PROCEDURE The PCR conditions and assay conditions described require precisely controlled conditions Deviations from these parameters may lead to product failure All instruments must be calibrated according to manufacturer s recommendations and oper
20. ry with nuclease free water Keep all samples at similar concentrations B DNA amplification PCR 1 Allow the Master Mix to warm to room temperature 18 to 30 C 2 Gently vortex for approximately 10 seconds This will ensure the salts are in solution Spin briefly 5 10 seconds in microcentrfuge to bring contents to the bottom of the tube 3 Using Table 1 below prepare the components for amplification for n 1 reactions using the indicated amount of each component per reaction except for DNA Bring to a final volume of 20uL per reaction with nuclease free water Gently vortex 4 Pipette the appropriate amount of Genomic DNA 40 to 120ng into the PCR tubes 5 Aliquot the amplification mix into the PCR tubes containing the genomic DNA The total volume of amplification mix and genomic DNA should equal 20uL for each sample reaction 6 Cap tubes tightly to prevent evaporation during PCR 7 Place samples in the thermal cycler and run program see Table 2 and 3 Page 4 of 9 LC1436RUO 4 05 15 Table 1 Reaction Components for Amplification Component Amount per PCR sample reaction LIFECODES Master Mix 6uL Genomic DNA 10 200ng pL Total of 80ng LIFECODES Tag Polymerase 0 2uL 1U Nuclease free water To 20uL final volume Table 2 Thermal Cycler Conditions for Amplification Thermal Cycler Mode Ramp speed MAX mode 3 9 C sec 9700 MAX mode 3 9 C sec GeneAmp PCR System 9700
21. teps The instrument can be turned off at this point if it is not going to be used for the remainder of the day 3 After a batch is complete the data is exported as a comma separated values csv file These files are named OUTPUT CSV and saved in a folder with the Batch Name This data is then available for making typing assignments as described below Refer to Luminex User s Manual for instrument operation including daily startup calibration maintenance and shutdown procedures RESULTS Sample typing can be done as follows The generated CSV files can be opened and the data processed with common spreadsheet programs such as Microsoft Excel Lotus 123 Corel Quattro Pro or similar software Analysis is comprised of the following steps 1 Verify that the Number of Events for each SSO in each sample is at least 60 This information is found in the DataType Count section of the CSV file 2 Determine that the values for the Consensus probes for each sample are above their minimum Median Fluorescent Intensity or MFI The minimum thresholds are lot specific and can be found in the Threshold Table Caution e To obtain reliable results there must be sufficient data gathered by the Luminex Instrument e Collect at least 60 events for HLA DQA1 Part 628933 and HLA DPB1 Part 628937 e Collect at least 40 events for HLA DQA1 B1 Part 628930R and HLA DPA1 B1 Part 628936R 3 Subtract the Background Control value for each probe
22. x system That is all SSOs are analyzed simultaneously and the entire assay is carried out in a single reaction vessel with the addition of a single reagent PRINCIPLES OF THE PROCEDURE The LIFECODES HLA SSO Typing procedure is based on the hybridization of labeled single stranded PCR product to SSO probes Amplification of DNA using PCR typically employs equimolar amounts of both forward and reverse primer to generate a double stranded DNA product However if the amount of one primer is in excess relative to the other the reaction will generate some single stranded DNA product in addition to double stranded product During the initial cycles of the LIFECODES amplification step double stranded DNA is generated Once the limiting primer is exhausted the remaining primer uses the double stranded product as a template for generation of single stranded DNA This method generates both double stranded and single stranded products that upon denaturation will both participate in the hybridization reaction Each of the different probes may be homologous to a sequence within the amplified DNA that is unique to an allele or group of alleles In other words these probes are designed so that each probe preferentially hybridizes to a complementary region that may or may not be present in the amplified DNA In addition the amplified DNA is also hybridized to one or more Consensus probes homologous to sequences present in all the alleles of a locus SSO Typing can be
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