Instructions for Use
Contents
1. Support and Contact Details Conexio Genomics Pty Ltd Or your local distributor 2 49 Buckingham Dr Wangara 6065 Western Australia Tel 61 422 863 227 email support conexio genomics com Skype conexiocgx Website http www conexio genomics com Conexio and HARPS are trademarks of Conexio 4 Pty Ltd HARPS is a registered trademark in some territories Page 19 of 19 For In Vitro Diagnostic Use2. refer to the appropriate instrument user s manual for detailed instructions and guidance 7 Editing and analysis of electropherograms The SBT Resolver kits were developed and validated using the Assign SBT and Assign ATF software developed by Conexio Genomics Pty Ltd Users are recommended to use Assign SBT 3 6 or 4 7 as these versions of the software utilise setting and reference files specifically designed for the SBT Resolver typing kits and HARPS For more details in relation to the operation of these software please refer to the applicable user manuals available for download on the Conexio Genomics website http www conexio genomics com The sequencing based typing data generated using the SBT Resolver typing kits should be analysed against the following Assign reference files which are provided by Conexio Genomics Assay Product Code Assign Reference File SBT Resolver HLA DRB3 AN PD11 0 0 DRB3 xml SBT Resolver HLA DRB4 AN PD12 0 0 DRB4 xml SBT Resolver HLA DRB5 AN PD13 0 0 DRBS5 xml Alternatively the sequencing data for all of the assays can be analysed against the D345 xml reference so long as the naming convention is such that each locus is analysed separately Page 12 of 19 For In Vitro Diagnostic Use Performance Characteristics Accuracy Panels of up to 57 samples from the UCLA International DNA Exchange proficiency testing program 2008 2010 used for internal testing for t
3. 7 F 20 C1 CG570 R 20 C1 GC209 F 20 C1 GAA309 R 20 C1 AC362 F 20 C1 GG572 R 20 C1 AC302 R 20 C1 TG539 R 20 RB 52 F 20 For In Vitro Diagnostic Use RB GG125 F 20 RB AA197 F 20 RB TT197 F 20 RB GT196 F 20 RB TA164 F 20 RB TT227 F 20 RB AT258 F 20 RB GC258 F 20 RB AT257 R 20 RB TT321 R 20 RB GT344 R 20 RB TG344 R 20 Self certified SBT Resolver typing kits HH PD3 2 2 20 SBT Resolver HLA C kit 20 and 50 tests HH PD3 2 2 50 PQ PD6 2 2 20 SBT Resolver HLA DQBI kit 20 and 50 tests PQ PD6 2 2 50 HH PD10 1 20 SBT Resolver HLA DPB1 kit 20 and 50 tests HH PD10 1 50 Self certified HARPS QB TA122 F 20 QB TA173 F 20 QB CT173 F 20 QB TA185 F 20 QB GG353 R 20 PB GC112 F 20 PB TAC121 F 20 PB GC194 F 20 PB AT251 R 20 PB AG341 R 20 PB GG341 R 20 Assign SBT software Self certified ASS G N SB T3 6 Product code CGX0036 ASSIGNssrv47 Product code CGX00470 C E 0197 C For Research Use Only SBT RESOLVER LC PD2 9 20 SBT Resolver HLA B57 kit 20 and 50 tests LC PD2 9 50 General Purpose Laboratory Reagents MgCl2 1 0 50 2mM MgCl MgCL 1 0 3000 SEQ BUF 2 0 400 5x Seq Rxn Buffer SEQ BUF 2 0 5000 EDTA 3 0 200 125mM EDTA pH8 0 EDTA 3 0 5000 Please contact your local distributor for further details RB GA196 F 20 RB CT257 R 20 QB CG353 R 20 PB GT313 R 20 Page 18 of 19 For In Vitro Diagnostic Use
4. CONEXIO SBT RESOLVER Instructions for Use PCR Amplification and Sequencing of the HLA DRB3 DRB4 and DRB5 Loci Version No 3 0 Issue Date March 2014 adel EC REP Conexio Genomics Pty Ltd Qarad bvba 2 49 Buckingham Dr Cipalstraat 3 Wangara 6065 B 2440 Geel Western Australia Belgium Australia Page 1 of 19 For In Vitro Diagnostic Use Contents PRIN CIP LE iisscsscatceissecccssessnnedoatsaanseSestasasecsaanestsdadcetassseasencousescassscssdassebestsncadesceaasceissasasasssvbaseacesseess 3 INTENDED USE csscscecavsscssssececsstisvsevsssdesvescesovsctsssessscseatesssasestssuctecsssebsdaccesevesesstenssecscdsssensscesivsncssesssaee 3 KIT COMPOSITION vssvssscccvssseisessssssecsatecssvsececsdesseteateesacscsvsssssecsssssesesveseveseseseusdacselessescsstesvesestsesesees 4 STORAGE REQUIREMENT ccsssssssssssssssssscsssssscssssssscccccsccscssssssnscsccscccccssssssssssssssccsscsscssssssssens 5 MATERIALS REAGENTS AND EQUIPMENT NOT SUPPLIED ccccccssssssssssssscccccsssssees 5 SAMPLE REQUIREMENTS iiccicciscsivcsvesssscecccvssocssessvessessucceccbetiessssdevsevsbevedtcssvssvessssdvessebencevessvostesests 6 WARNINGS AND SAFETY PRECAUTIONS ccsssssssssssscccsscsssssssssscssccccccssccssssssssccccesccesesssssens 7 SYMBOES secsscsscccascstssnececiescnsvssesenssesseavseuscecsevennesdesenscasdcndsentesscnesnscedboansaesesdsurssevsondandssedesssscsennansesedesees 7 PROCEDUR Eoissasiadiscissacccsssscetestaasasecssstasceedes
5. Consider high Presence of high using a higher dilution factor fluorescent peaks following PCR purification artefacts Check the amount of DNA polymerase used in the PCR Too much reaction products Check instrument parameters applied to sequencer Consider reducing the injection time and voltage Noisy baseline high Contaminated PCR product Refer to corrective actions background listed above Amplification of closely related HLA genes Check parameters thermal cycling Poor PCR purification Ensure ExoSAP IT treatment is undertaken according to kit s user instructions Ensure that the PCR mixture is mixed thoroughly with ExoSAP IT Consider using Exo SAP IT following the manufacturer s procedure increasing the amount of enzyme or consider an alternative purification technique Contaminated reactions sequencing Ensure that all steps are taken to prevent cross contamination Change pipette tips wherever possible Add liquids at the top of the reaction wells Prevent aerosols Page 16 of 19 For In Vitro Diagnostic Use Problem Possible cause s Solution Contaminated sequencing Check sequence quality of the primer other sequencing primers and other samples using the same primer Consider using a fresh aliquot of sequencing primer Contaminated dye terminator Repeat sequencing with fresh mix or sequencing buffer aliquot of re
6. agents Poor purification of sequencing Repeat sequencing and ensure products that purification is undertaken according to manufacturer s instructions Presence of Dye blobs Poor purification of sequencing Purify products according to products kit instructions Ensure products are washed sufficiently with 80 ethanol Related Products CE marked IVDs SBT RESOLVER XH PD1 1 2 20 XH PD1 1 2 50 BS PD2 1 2 20 BS PD2 1 2 50 HH PDS 2 5 20 HH PDS 2 5 50 SBT Resolver HLA A kit 20 and 50 tests SBT Resolver HLA B kit 20 and 50 tests SBT Resolver HLA DRB1 kit 20 and 50 tests SBT RESOLVERuares C1 TT98 F 20 C1 CT102 F 20 C1 GG307 R 20 CI TA368 F 20 C1 BTA F 20 C1 GA206 F 20 C1 AG360 F 20 C1 CC486 F 20 C1 CG572 R 20 C1 CT97 F 20 C1 GC302 R 20 C1 GG539 R 20 RB 01 F 20 C1 AC98 F 20 C1 CC102 F 20 C1 GG363 AF 20 C1 GT355 R 20 C1 BCG F 20 C1 CG319 F 20 C1 GC363 F 20 C1 CT559 R 20 C1 GAG601 R 20 CI CT112 F 20 C1 CG343 F 20 C1 AA601 R 20 RB 04 F 20 C1 TC98 F 20 C1 AG203 F 20 C1 TA363 F 20 C1 GG362 R 20 C1 CC144 F 20 C1 CA309 R 20 C1 GG363 BF 20 C1 GA559 R 20 C1 CG134 F 20 C1 CA343 F 20 C1 AG595 R 20 RB 09 F 20 Page 17 of 19 C1 TA98 F 20 C1 GT240 F 20 C1 AT362 F 20 CI CT423 F 20 C1 AC206 F 20 CI GAT309 R 20 C1 TA420 F 20 CI AC559 R 20 C1 AG270 F 20 C1 GA361 F 20 RB 15 F 20 C1 CA102 F 20 C1 TT368 F 20 C1 AC49
7. aker PCR products may require a lower dilution factor 3 3 ExoSAP IT treated samples may be stored at 4 C until ready for use ExoSAP IT treated samples can be stored at 4 C for up to a week before use but should be stored at 20 C for long term storage 4 Sequencing Reaction NOTE Only HLA DRB3 DRB4 and DRBS5 positive samples identified by gel electrophoresis should be sequenced using the following procedure Page 9 of 19 For In Vitro Diagnostic Use 4 1 Table 3 lists the sequencing primers that are to be used for each locus Locus Sequencing Primers HLA DRBS DRB5EX2F DRB5EX2R DRB5EX3F Table 3 Sequencing primers provided to sequence the positive samples for each locus 4 2 Prepare a fresh solution of sequencing primer mix on ice each time a sequence reaction is performed The composition and volumes for the mix are indicated per sample Component Volume Sequencing primer 2 uL Sterile water 11 5 uL BigDye Terminators l uL 5X Sequencing buffer 3 5 uL 4 3 Mix each sequencing reaction mix gently by pulse vortexing 4 4 Dispense 18uL of the sequencing reaction mix to each appropriate reaction tube well 4 5 Add 2uL of purified PCR product to each appropriate well NOTE Care must be taken to prevent cross contamination of sequence reactions 4 6 Seal the reaction tubes mix gently and centrifuge briefly to ensure that the contents are located at the base of each reaction tube 4 7 Place the
8. amples whose HLA type has been determined by other molecular based procedures In particular any deviations from this procedure e g the use of alternative PCR or DNA sequencing purification procedures must be validated by the user prior to implementation e These kits have been validated using panels of samples whose genotypes cover a broad range of alleles However it should be noted that rare alleles and alleles with polymorphisms in amplification and sequencing primer sites may be encountered and these may not be amplified or sequenced Page 13 of 19 For In Vitro Diagnostic Use A positive control human DNA sample known to have HLA DRB3 DRB4 DRBS5 a negative control human DNA sample known to be negative for HLA DRB3 DRB4 DRB5 and no template control sterile water must be included on every PCR run The positive control must produce two amplicons of the appropriate size and the resultant sequence must be in concordance with the sample s genotype The negative control must produce a single internal control amplicon of the appropriate size There must be no PCR products in the no template control for each experiment If a band is evident contamination may have occurred at some level and the run must be repeated Occasionally there may be larger fainter PCR products evident These additional bands do not interfere with sequence results or quality License The SBT Resolver kits contain GoTaq Hot Start Polymerase DNA POL which
9. ate control reaction well 1 6 Seal the reaction wells Mix gently by vortexing and centrifuge briefly 1 7 Place the reaction wells into a thermal cycler and amplify the target sequence according to the thermal cycling conditions below 95 C 10 mins 96 C 20 secs 60 C 30 secs 33 cycles 72 C 3 mins 15 C hold 1 8 Amplification takes approximately 2 5 hours to complete 1 9 When the PCR is completed remove the plate from the thermal cycler and either proceed directly to gel electrophoresis or store at 4 C until required NOTE Purification of positive amplicons by ExoSAP IT treatment should occur within 24 hours of completion of PCR 2 Agarose Gel Electrophoresis 2 1 Confirm successful amplification of the internal control amplicon in for all DNA samples tested and the applicable HLA DRB3 DRB4 and DRBS5 target amplicons Page 8 of 19 For In Vitro Diagnostic Use in positive control and positive DNA samples by agarose gel electrophoresis using SuL of each PCR product combined with 5uL of loading buffer alternative volumes of loading buffer should be validated prior to use The use of 1 agarose gels is recommended 2 2 All samples tested using the HLA DRB3 DRB4 and DRB5 PCR mixes should amplify the internal control amplicon regardless of the HLA DRB3 DRB4 DRB5 genotype Positive samples should amplify both the internal control amplicon plus the target amplicon The expected sizes of each amplic
10. cyclers MJ Research PTC 225 DNA Engine DYAD Applied Biosystems by Life Technologies GeneAmp PCR System 9700 and Eppendorf Mastercycler Pro Use of other thermal cyclers with these kits requires validation by the user 4 0 2mL thin walled thermal cycling reaction tubes 8 well strips or 96 well plates Use those recommended for use with your thermal cycler 5 Sterile 1 5mL tubes 6 Sterile biological safety cabinet or hood T Table top centrifuge with plate adapters and capacity to reach 2500 x g 8 Vortex Agarose Gel Electrophoresis 9 Agarose gel electrophoresis apparatus 10 1 agarose molecular biology grade TBE gel containing 0 lug mL ethidium bromide 11 Loading buffer 12 PCR Marker suitable to cover range of 300 1300 bp 13 UV transilluminator Purification of PCR Product 14 ExoSAP IT USB Products Cat No 78200 for 100 reactions Page 5 of 19 For In Vitro Diagnostic Use 15 2mM MgCl available for purchase from Conexio Genomics product code MgCl2 1 0 50 or MgCI2 1 0 3000 16 Shaker The use of alternative PCR purification techniques requires validation by the user prior to use Sequencing Reaction 17 BigDye Terminator Cycle Sequencing Kit v3 1 or v1 1 Applied Biosystems by Life Technologies 18 5x Sequencing Reaction Buffer Conexio Genomics product code SEQ BUF 2 0 400 or SEQ BUF 2 0 5000 or BigDye Terminator v3 1 or v1 1 5X Sequencing Buffer Applied Bios
11. e 6 of 19 For In Vitro Diagnostic Use Uae and Safety Precautions This kit must be used by trained and authorized laboratory personnel All samples equipment and reagents must be handled in accordance with good laboratory practice In particular all biological material should be considered as potentially infectious The use of gloves and laboratory coats is strongly recommended Handle and dispose of all sample material according to local and national regulatory guidelines There are NO dangerous substances contained in any of the kit components Do NOT use reagents beyond their expiration date The use of kit components from different kit batches is NOT recommended Such use may affect the assay s performance Use of reagents not included in this kit or not listed under Materials Reagents and Equipment Not Supplied e g alternative DNA polymerases is NOT recommended Such use may affect the performance of the assay Care should be taken to prevent cross contamination of DNA specimens Change tips between DNA specimens wherever possible Pre and Post PCR activities must be strictly physically separated Use specifically designated equipment reagents and laboratory coats Ethidium bromide is a potential carcinogen Protective gloves must always be used when preparing and handling gels Dispose of ethidium bromide gels and buffers according to local and national guidelines While viewing and photographing agarose gels unde
12. from Conexio Genomics Pty Ltd Intended Use These Conexio Genomics SBT Resolver SBT kits are used for the typing of the HLA Class II genes HLA DRB3 DRB4 and DRB5 in a laboratory setting from genomic DNA Each kit contains reagents that facilitate the PCR amplification and DNA sequencing of a given gene The resultant sequencing data is then interpreted through the use of Conexio Genomics Assign SBT software It should be noted that these kits are not to be used for the diagnosis of disease Page 3 of 19 For In Vitro Diagnostic Use Kit Composition Kit Catalogue No 17 PRE PCR Contents POST PCR Contents No of vials No of vials Locus HLA DRB3 AN PD11 0 0 20 20 tests 1 x 10uL 1 x 44uL each HLA DRB3 MIX 1 x 370uL AN PD11 0 0 50 50 tests 1 x 20uL 1 x 110uL each HLA DRB3 MIX 1 x 920uL HLA DRB4 AN PD12 0 0 20 20 tests E 1x 10uL 1x 44uL each HLA DRB4 MIX 1 x370uL AN PD12 0 0 50 50 tests E 1 x 20L 1 x 110uL each HLA DRB4 MIX 1 x 920uL HLA DRB5 AN PD13 0 0 20 20 tests E 1x 10uL 1 x 44uL each HLA DRB5 MIX 1 x 370uL AN PD13 0 0 50 50 tests E 1 x 20uL 1 x 110uL each HLA DRB5 MIX 1 x 920uL o g g iw iw zZ lt gt gt gt gt gt gt UV UV p UV UV UV O O O O ie m m m m m a o o o iw iw JJ JJ es JJ JJ JJ w w W W is ja a a E A o W The PRE PCR component of each kit consists of a vialof a locus specific PCR mi
13. he SBT Resolver kits yielded the following results Locus Number Number of Number of Numberof Number of of samples samples mistyped heterozygous unique tested containing samples samples alleles target locus HLA DRB3 54 22 0 0 3 HLA DRB4 56 33 0 1 HLA DRBS5 57 15 0 0 4 Sequence analysis of PCR and sequencing primer sites and performance evaluation has not identified any common and well documented alleles that are not amplified through the recommended use of these kits Detection Limit The recommended concentration of high molecular weight human genomic DNA is 20 100ng uL Internal testing has shown that samples with concentrations as low as 5ng uL can also be used Correct genotypes were also obtained from poor quality or sheared DNA Specificity Conexio Genomics Pty Ltd s SBT Resolver kits are locus specific assays Use of the kits according to these instructions should only amplify a single locus In most instances the use of the sequencing primers incorporated in each kit will produce a HLA typing for most samples without the need for further resolution It should be noted that mutations at amplification or sequencing primer sites are possible and may result in allele drop out Samples that suggest a homozygous typing result must be confirmed by alternative procedures Limitations and Cautions e It is strongly recommended that these kits are validated by the user prior to implementation in the laboratory using s
14. he reaction tubes at 98 C for 5 minutes Following incubation ensure that the reaction wells are cooled quickly to room temperature e g place on ice or use the thermal cycler to perform the denaturation and cooling steps before being placed on the sequencer If it is not possible to run the plates immediately store at 4 C until required NOTE ENSURE THAT THERE ARE NO AIR BUBBLES IN THE REACTION WELLS THESE CAN ENTER AND DAMAGE THE CAPILLARY Page 11 of 19 For In Vitro Diagnostic Use 6 3 Load the reaction plate onto the automated sequencer and prepare the data collection file according to the sequencer manufacturer specifications 6 4 The following instrument parameters have been validated by the manufacturer using Big Dye Terminator Sequencing Kit v3 1 and POP 7 These parameters may require user validation for other polymers sequencing chemistries and instruments Please refer to the appropriate instrument user s manual for detailed instructions and guidance e g ensure that the dye set setting is appropriate for the chemistry used for example v1 1 Big Dye Terminator sequencing chemistry will require a different dye set Parameter Setting Dye set Z_BigDyeV3 Mobility file KB_3730_POP7_BDTV3 Basecaller KB bcp Run Module Regular FastSeq50_POP7 Injection time 15 sec Collection time 3000 sec 6 5 Use the instrument s data collection software to process the raw collected data and create the sequence files Please
15. he seals to the reaction tubes and discard the supernatant by inverting the reaction tubes onto paper towel or tissues 5 7 Place the inverted reaction tubes and paper towel or tissue into the centrifuge Centrifuge at 350g for 1 minute to remove any residual supernatant 5 8 Remove the reaction tubes from the centrifuge and replace in an upright position on the work bench Discard the paper towel or tissues 5 9 Prepare a fresh solution of 80 ethanol with absolute ethanol and sterile water 5 10 Add 60uL of 80 ethanol to each reaction tube well Reseal the tubes and mix by vortexing briefly 5 11 Spin at 2000g for 5 minutes 5 12 Repeat steps 5 6 to 5 7 5 13 Remove the reaction tubes from the centrifuge and discard the paper towel Reseal the reaction tubes and proceed to the denaturation step Otherwise store at 20 C in the dark It is recommended that the extension products are run on the DNA sequencer within 24 hours of setting up the sequencing reactions Denaturation amp Electrophoresis of Sequencing Reaction Products NOTE The procedure for the denaturation of extension products in Hi Di Formamide described here may not be necessary if purification procedures other than the ethanol precipitation have been used It is strongly recommended that users validate alternative procedures before proceeding 6 1 Add 12uL of Hi Di Formamide to each reaction tube Vortex and centrifuge the tubes briefly 6 2 Incubate t
16. is manufactured by Promega Corporation for distribution by Conexio Genomics Pty Ltd Licensed to Promega under U S Patent Nos 5 338 671 and 5 587 287 and their corresponding foreign patents Page 14 of 19 For In Vitro Diagnostic Use Troubleshooting Problem Possible cause s Solution No or weak PCR product Poor quality DNA Assess DNA quality by gel electrophoresis Intact DNA should be approx 3kb with little or no evidence of smearing on gel Re extract DNA and repeat PCR where possible Insufficient quantity of DNA added to PCR Check concentration of DNA is between 20 100ng uL Re extract DNA and repeat PCR where possible Presence of PCR inhibitors in genomic DNA Avoid the use of whole blood specimens containing heparin Re extract DNA and repeat PCR where possible DNA polymerase not added to Repeat PCR Ensure the mastermix or insufficient mastermix components are mixing of mastermix prior to addition to samples added and mixed sufficiently by vortexing Thermal cycling problems Check the thermal cycling run parameters Check the run history to ensure that the run was not terminated prematurely Ensure that the thermal cycler is operating according to manufacturer s specifications and is regularly maintained No ethidium bromide added to the gel Submerge the gel in a staining bath containing 1X TBE with 0 5mg mL ethidium bromide Destain in 1X TBE bef
17. on are listed in Table 2 Locus Expected band sizes HLA DRB3 target amplicon 640 bp HLA DRB4 target amplicon 460 bp HLA DRBS target amplicon 470 bp Internal control band 400 bp Table 2 Expected product sizes for each assay 3 Purification of PCR Product NOTE Purification systems other than ExoSAP IT e g ExoSTAR Agencourt AMPure XP or column based systems can be used to purify these PCR products It is strongly recommended that users validate these procedures before proceeding If ExoSAP IT is to be used it is recommended that users follow the procedure described below 3 1 Prepare a mastermix consisting of 4uL of ExoSAP IT and 8uL of 2mM MgCl per sample Gently pulse vortex to mix Dispense 12uL of the mastermix into the reaction well of each positive sample Seal the tubes vortex and place on a shaker or gently vortex for 2 mins Centrifuge briefly before placing into the thermal cycler Run the thermal cycler according to the following profile 37 C 30mins 80 C 15mins 4 C hold 3 2 Upon completion dilute the purified product 1 4 with sterile water This dilution step will ensure that there is sufficient template to perform the sequencing reactions and ensure that the concentration of the template is sufficient to produce good quality sequence data NOTE A higher dilution factor e g 1 8 may be required if consistently high signals and associated noise and artefacts are observed We
18. ore taking gel image Ensure ethidium bromide is added to gel prior to pouring DNA samples are eluted or diluted in water that can have a slightly acidic pH Wherever possible use sterile water with a neutral pH Incorrect band sizes Incorrect kit used Check that the appropriate kit has been used Incorrect thermal program used cycling Check the parameters thermal cycle PCR contamination Check the negative control for evidence of contamination Decontaminate work area and repeat PCR Repeat PCR to identify source of contamination Consider using a fresh kit Page 15 of 19 For In Vitro Diagnostic Use Problem Possible cause s Solution If the genomic DNA of a sample appears to be contaminated re extract or obtain an alternative source of DNA Weak signal intensity of electropherograms Weak PCR product Check gel image Sequencing weak PCR bands is NOT recommended as the sequence quality may be insufficient for SBT Consider using a lower dilution factor eg 1 2 1 3 after PCR purification Insufficient reaction products applied to sequencer Check sequencer parameters Injection time and voltage may need to be increased Problems during purification of sequencer products Use extreme care when discarding the supernatant as it may dislodge the pellet Signal intensity is too Too much PCR product Check the gel image
19. r UV light always avoid direct exposure and use appropriate UV blocking face protection disposable gloves and laboratory coats Symbols The following non standard symbols have been used Symbol Description HLA X MIX Locus specific PCR Mix ae DRB3EX2F HLA DRB3 exon 3 forward sequencing primer Refer to Kit Composition and Table 3 for other sequencing primers Date of manufacture required for non EU markets Page 7 of 19 For In Vitro Diagnostic Use Procedure 1 PCR 1 1 Set up one reaction for each sample for each locus being amplified Include appropriate positive and negative amplification controls of known genotype and at least one no template control for each group of samples being amplified 1 2 Prepare a fresh solution of PCR master mix each time a PCR is performed Quickly thaw the required number of vials of the appropriate PCR Mix Once thawed vortex briefly 1 3 Dispense the required amount of PCR mix and DNA polymerase into a sterile tube for the number of samples to be tested Refer to Table 1 below Pulse vortex the solution 3 4 times Locus DRB3 Locus specific PCR Mix 16 7uL e g HLA DRB3 MIX DNA Polymerase 0 3uL e 8 DNA POL DRB3 Table 1 Composition of the master mix required per sample 1 4 Dispense 17uL of the master mix into each reaction well 1 5 Add 3uL of sample DNA or appropriate control sample to each reaction well Add 3uL of sterile water to the no templ
20. reaction tubes into a thermal cycler and run according to the following profile Number of cycles Temperature and time 25 96 C 10sec 50 C 5sec 60 C 2min 1 4 C hold 4 8 Once the program is complete remove the reaction tubes from the thermal cycler and either proceed directly to purification of the reaction products or store at 4 C until required It is recommended that samples are purified and run on the DNA sequencer within 24 hours 5 Purification of Sequencing Reaction Products NOTE Purification of the reaction products may be carried out by procedures other than the ethanol precipitation method described here It is strongly recommended that users validate these procedures before proceeding Page 10 of 19 For In Vitro Diagnostic Use 5 1 Briefly centrifuge the reaction wells plates before proceeding If reusable lids caps have been used during thermal cycling label the lids caps to avoid cross contamination 5 2 Carefully remove the seal 5 3 To each reaction tube add SuL of 125mM EDTA pH8 0 Ensure that the EDTA reaches the base of the reaction tube 5 4 Add 60 uL of 100 ethanol to each reaction well Seal the plate and vortex briefly but thoroughly to ensure thorough mixing 5 5 Pellet the extension products by centrifuging at 2000g for 45 minutes IMMEDIATELY PROCEED TO THE NEXT STEP If this is not possible re centrifuge for an additional 10 minutes before proceeding 5 6 Remove t
21. scdnnsscecvedenscsstecdsacsccvanstevssvevacsessnscsedevedectessiansseevestesseactentencdevsess 17 SUPPORT AND CONTACT DETAILS cccsssssssssrcrcccccccccssssssnscsecccccsscccesesssssssccccsccceesescses 19 Page 2 of 19 For In Vitro Diagnostic Use Principle The HLA Sequencing Based Typing SBT procedure described here involves the initial locus specific PCR amplification of the HLA DRB3 DRB4 and DRB5 genes Presence of target and the internal control amplicons are visualised by agarose gel electrophoresis Samples that are positive for the HLA DRB3 DRB4 or DRB5 genes will produce two amplicons the target amplicon and internal control while negative samples will produce a single amplicon only internal control Positive samples are then further characterised by DNA sequencing following treatment with ExoSAP IT to remove unincorporated primers and dNTPs The target amplicon is then used as a template for direct automated fluorescent DNA sequencing using customized sequencing primers and the BigDye Terminator sequencing chemistry available from Applied Biosystems by Life Technologies The extension products are purified according to the ethanol precipitation method and denatured using Hi Di formamide available from Applied Biosystems by Life Technologies before separation and detection on an automated fluorescent DNA sequencer It is recommended that the resulting data is analysed with Assign SBT sequence analysis software
22. vaanesdesactenstaseansacessavensaaschaeanachestsnnacucseauacessesesssassedasessesceaes 8 1 PER enter erry ars Toa RLL en Tanga EAA ASA A ceritreeee nacre rr eer rere 8 2 AGAROSE GEL ELECTROPHORESIS sssssssesssecceecccceccceeeeeeeaaeeeaeeeseesesseeeeseeeeceeeceeeeeeeseeeeeaaesagaaga 8 3 PURIFICATION OF PER PRODUC T aesssessoeszeses aa e a rea a aa a a EOE TEP a Santa ad EIE load 9 4 SEQUENCING REACTION naren en a a a a a T E a 9 5 PURIFICATION OF SEQUENCING REACTION PRODUCTS ss ssssssesescsseecceecceeeecesseeeseaaeeaaesseenes 10 6 DENATURATION amp ELECTROPHORESIS OF SEQUENCING REACTION PRODUCTS s0 ssseeseeees 11 7 EDITING AND ANALYSIS OF ELECTROPHEROGRAMS ccccccscesseeessesssseeeeeeecececesssseseaeaaeceeeeeeeees 12 PERFORMANCE CHARACTERISTICS eseeeeesesseesssssssecccccsssssscsssccecccccssssssocccccececceessssssececcceeeeee 13 ACCURACY EEE AA E E ET E EEA EE EE EAE AE AEE 13 TDETHO TION LIMIT PE REAA AER AEA A REC A A A AEAT 13 SPEGIPICIT e E E E A E EDS DA SEUS IPod aa ER 13 LIMITATIONS AND CAUTIONS sssssssssssssscsssssssssssssssssscccccccssssssnsssccccccccssscscsssssscccscsccesessseses 13 EICENSE sicscsvsssscscescesevssesssonsxecscssvsccssevetvctevseesecassvessedeosesseacesssceibextesesenesvesedenssstessatecsevsesscsesdessessesess 14 TROUBLESHOOTING ovsssccscsssessvsevsstvectacsovssscccdsvesnccsussvssstesdessescasssdeseasesevsonsssissdecsasevsencssessdansvesssess 15 RELATED PRODUCTS iieivcincccctennsicse
23. x e g consisting of PCR buffer dNTPs MgCl locus specific PCR primers and a single vial of DNA polymerase e g The POST PCR kit contains sequencing primers e g Page 4 of 19 For In Vitro Diagnostic Use Storage Requirements The PRE and POST PCR boxes may be separated and stored in designated PRE and POST PCR freezers When stored at 20 C temperature range of 15 C to 25 C is acceptable the kit components can be used until the expiry indicated on the outer kit containers and can tolerate up to 25 freeze thaw cycles Accelerated stability testing for these kits have indicated a shelf life of two and a half years from the date of manufacture when stored at 20 C While confirmatory real time testing is underway it is strongly recommended that these kits are NOT to be used beyond their expiry date To maintain optimal kit performance the kit components should be removed from the 20 C storage location and thawed rapidly at room temperature before use The kit components with the exception of the polymerase should then be gently vortexed to ensure that the components of each tube are appropriately mixed after thawing After use the kits components should be returned immediately to 20 C Materials Reagents and Equipment Not Supplied PCR 1 Sterile water 2 Electronic or mechanical pipettes and aerosol resistant tips 3 Thermal cycler with heated lid These kits have been tested using the following thermal
24. ystems by Life Technologies Purification of Sequencing Reaction Products 19 125mM EDTA pH8 0 available for purchase from Conexio Genomics product code EDTA 3 0 200 or EDTA 3 0 5000 20 Absolute Ethanol Each run needs freshly prepared 80 ethanol solution consisting of absolute ethanol and sterile water DO NOT USE DENATURED ETHANOL The use of alternative sequencing purification techniques requires validation by the user prior to use Denaturation and Electrophoresis of Sequencing Reaction Products 21 Hi Di Formamide Applied Biosystems by Life Technologies product code 4311320 22 Automated DNA Sequencer and accessories eg Applied Biosystems by Life Technologies ABI Prism 3730 including data collection and software These kits have been tested and validated on the Applied Biosystems by Life Technologies 3100 3730 and 3730x1 capillary sequencers and software The use of other denaturation techniques and sequencing platforms requires validation by the user prior to use 23 HLA Sequencing Analysis Software e g Assign SBT version 3 6 or higher Conexio Genomics Pty Ltd Sample Requirements 1 Sterile water negative no template control 2 High molecular weight human genomic DNA concentration range of 20 100ng uL in Tris EDTA buffer and ODz6o280 gt 1 8 extracted from ACD or EDTA anticoagulated whole blood specimens Do NOT use whole blood specimens containing heparin Pag
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