Instructions for Use
Contents
1. SBT RESOLVER Instructions for Use PCR Amplification and Sequencing of HLA Class I and II Loci Version No 15 0 Issue Date August 2015 Conexio Genomics Pty Ltd Qarad bvba 2 49 Buckingham Dr Cipalstraat 3 Wangara 6065 B 2440 Geel Western Australia Belgium Australia Page 1 of 25 For Diagnostic Use Contents Le Eg P oie PN 3 INTENDED USE OEC 3 KIT COMPONSITIQN ees cossecccedssseckessaecbuassesens eU seen suec pe ve sk eveseebucssesess 4 STORAGE REQUIREMENTS eseesseceesseceesseceessceceessccecsscceesseceesscceesseceessececsseceesseceesseceessosee 9 MATERIALS REAGENTS AND EQUIPMENT NOT SUPPLIED e eene eren 9 SAMPLE REQUIREMENTIS 3 etis re aeo asaini iiaa 10 WARNINGS AND SAFETY PRECAUTIONS ee ee eere ee eee tone etta tn esee te enne ette ener een 11 dip EC 11 DERE 12 1 PGR PLC 12 2 AGAROSE GELELECTROPHORESIS idee sade ates e Ege te cave dr vea Rede eee 12 3 PURIFICATION OF PCER PRODUCT oe erre ento Sere eee ee sede teer ex ene Peter eva ee ee eode cheeses 13 4 SEQUENCING REACTION 14 5 PURIFICATION OF SEQUENCING REACTION 15 6 DENATURATION amp ELECTROPHORESIS OF SEQUENCING REACTION 16 7 EDITING AND ANALYSIS OF ELECTROP2. 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 validated using the following thermal cyclers MJ Research PTC 225 DNA Engine DYAD Applied Biosystems by Life Technologies Veriti Thermal cycler Gene Amp 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 Sterile 1 5mL tubes Sterile work area such as biological safety cabinet or hood Table top centrifuge with plate adapters and capacity to reach 2500 x g Coe cl Se A cn Vortex Agarose Gel Electrophoresis 9 Agarose gel electrophoresis apparatus 10 1 agarose molecular biology grade TBE gel containing O 1ug 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 USB ExoSAP IT Cat No 78200 for 100 reactions or ExoProStar Cat No US77702 for 100 reactions Page 9 of 25 For In Vitro Diagnostic Use 15 16 2mM MgCl Available for purchase from Conexio Genomics product code MgCI2 1 0 50 or MgCI2 1 0 3000 Shaker The use of alternative PCR purification techniques
3. 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 No or weak PCR product for the exon 3 5 band for the KD PD10 2 1 assay Poor quality DNA Amplification of samples of very poor quality may result in weak amplification of the exon 3 5 amplicon Typing can still be achieved using exon 1 and 2 sequence data Alternatively re extract DNA and repeat PCR where possible Incorrect band sizes Incorrect kit used Check that the appropriate kit is used Incorrect thermal program used cycling Check the parameters thermal cycle Page 21 of 25 For Diagnostic Use 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 If the genomic DNA of a sample appears to 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 lower dilution factor e g 1 2 1 3 after PCR purification Insufficient reaction products applied to sequencer Check sequencer parameters Injection
4. Self certified 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 AN PD6 2 3 20 AN PD6 2 3 50 HH PD10 1 20 SBT Resolver HLA DPBI kit 20 and 50 tests HH PD10 1 50 KD PD10 2 1 20 KD PD10 2 1 50 Support and Contact Details Conexio Genomics Pty Ltd Or your local distributor PO Box 1294 Fremantle 6959 Western Australia Australia Tel 61 08 9336 4212 email support conexio genomics com Skype conexiocgx Website www conexio genomics com For ordering details please refer to the Olerup website http www olerup com Conexio and HARPS are trademarks of Conexio 4 Pty Ltd HARPS is a registered trademark in some territories Page 25 of 25 For In Vitro Diagnostic Use
5. 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 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 seals 5 3 To each reaction well add SuL of 125mM EDTA pH8 0 Ensure that the EDTA reaches the base of the reaction well 5 4 Add 60uL of 100 ethanol to each reaction well Seal the wells plate and vortex briefly but thoroughly to ensure thorough mixing Page 15 of 25 For In Vitro Diagnostic Use 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 the seals to the reaction wells and discard the supernatant by inverting the reaction wells onto paper towel or tissues 5 7 Place the inverted reaction wells and paper towel or tissue into the centrifuge Centrifuge at 350g for 1 minute to remove any residual supernatant 5 8 Remove the reaction wells from the centrifuge and place in an upright position on the work bench Discard the paper towel or tissues 5 9 Prepare fresh solution of 8096 etha
6. 38 100 100 0 34 15 AN PD6 2 3 HLA DPBI 77 100 100 0 60 18 HH PD10 1 HLA DPBI 16 10096 100 2 14 13 KD PD10 2 1 The two discordant samples contained additional sequence information outside exon 2 that was not reported by the UCLA International DNA Exchange proficiency testing program One sample contained 131 01 but was reported as 13 01 by the UCLA International DNA Exchange proficiency testing program The alleles differ in exons 3 and 4 The other sample contained 107 01 but was reported as 13 01 by the UCLA International DNA Exchange proficiency testing program These alleles differ in exon 1 For the SBT Resolver HLA DRB1 kits product code LG PD5 2 7 a panel of 23 well characterised samples covering a broad range of alleles was used for internal testing In addition a panel of 293 externally sourced samples were also typed without a priori knowledge of other HLA typing data These samples were also tested with the SBT Resolver HLA DQBI assay PQ PD6 2 2 In those cases where a homozygous result was obtained the DQB1 associations for those samples were examined to confirm result as well as to detect instances where allele drop out may have occurred The testing yielded the following results Locus Number Diagnostic Diagnostic Number Number of Number of sensitivity specificity of heterozygous of unique samples discordant samples alleles tested samples HLA DRBI 23 10096 10096 0 23 12 286 97 9
7. 50 tests 1 x 60uL 1 x 110uL each Page 4 of 25 For In Vitro Diagnostic Use Kit Catalogue No PRE PCR Contents POST PCR Contents No of vials No of vials HLA C HH PD 32 200 20 tests 1x 25 1 x 44uL each HH PD 3 2 2 50 50 tests 1 x 60uL 1 x 110uL each Class II HLA DRBI 5 2 5 20 20 tests 1 x 100 DRB1EX2R 2 1 x 44uL each RB TG344 R HLA DRB1 MIX 1x 370uL DRB1EXSR 2 Page 5 of 25 For In Vitro Diagnostic Use Kit Catalogue No b PRE PCR Contents POST PCR Contents No of vials No of vials 5 2 5 50 50 tests DNA POL DRB1 1 x 20uL 1x 110uL LG PD5 2 7 20 20 tests 1 x 10pL 1x 44uL each RB TG344 R LG PD5 2 7 50 50 tests DNA POL DRB1 1 x 20uL 1x 110uL HLA DQBI PQ PD62 200 20tests 1 x 10pL 1 x 4AuL each PQ PD6 2 2 50 50tests 1 x 20uL 1x 110pL each each Page 6 of 25 For In Vitro Diagnostic Use Kit Catalogue No ES PRE PCR Contents POST PCR Contents No of vials No of vials AN PD6 2 3 50 50 tests DNA POL DQB1 1 x 20uL 1 x 110uL each HLA DPBI HH PD10 1 20 20 tests 1x 10uL 1x 44uL each each KD PD10 2 1 20 20 tests DNA POL DPB1 1 x 10uL 1 x 44uL each Page 7 of 25 For In Vitro Diagnostic Use Kit Catalogue No PRE PCR Contents POST PCR Contents No of vials No of vials KD PD10 2 1 50 50 tests 1 x 20uL DPB1EX1F D
8. 99 6 0 253 33 Page 18 of 25 For In Vitro Diagnostic Use Six samples failed to amplify due to poor quality DNA samples One sample was found to contain contaminating DNA the source of which occurred at the laboratory from which the samples were obtained As a result of the contamination a genotype could not be obtained for that sample Sequence analysis of PCR and sequencing primer sites and performance evaluation studies have not identified any common and well documented alleles that have not been amplified through the recommended use of these kits For further information refer to the SBT Resolver Primer Analysis document available with each Assign SBT reference release downloadable from the Conexio Genomics website http www conexio genomics com 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 In those instances where heterozygous ambiguities remain the use of resolving seque
9. is then used as a template for direct automated fluorescent DNA sequencing using customized sequencing primers and the Big Dye 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 then analysed with Assign SBT sequence analysis software from Conexio Genomics Pty Ltd Intended Use Conexio Genomics SBT Resolver HLA SBT kits are used for the typing of HLA Class I HLA A B and C and Class II HLA DRB1 DQB1 and DPB1 genes 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 SBT kits are not used for the diagnosis of disease Page 3 of 25 For Diagnostic Use Kit Composition Kit Catalogue No PRE PCR Contents POST PCR Contents No of vials No of vials Class I HLA A XH PD1 1 2 20 20 tests 1x 25uL 1 x 44uL each XH PD1 1 2 50 50 tests 1 x 60uL 1 x 110uL each HLA B BS PD2 1 2 20 20 tests 1x25uL 1 x 44uL each BS PD2 1 2 50
10. requires validation by the user prior to use Sequencing Reaction 17 18 BigDye Terminator Cycle Sequencing Kit v3 1 or v1 1 Applied Biosystems by Life Technologies M 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 vl 5X Sequencing Buffer Applied Biosystems by Life Technologies Purification of Sequencing Reaction Products 19 20 125mM EDTA pH8 0 Available for purchase from Conexio Genomics product code EDTA 3 0 200 or EDTA 3 0 5000 Absolute and 80 Ethanol Each run requires freshly prepared 80 ethanol consisting of absolute ethanol and sterile water DO NOT USE DENATURED ETHANOL also known as methylated spirits in some countries The use of alternative sequencing purification techniques requires validation by the user prior to use Denaturation and Electrophoresis of Sequencing Reaction Products 2 22 23 Hi Di Formamide Applied Biosystems by Life Technologies product code 4311320 Automated DNA Sequencer and accessories e g 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 3730xl capillary sequencers and software The use of other denaturation techniques and sequencing platforms requires validation by the user prior to
11. resultant sequence must be in concordance with the sample s genotype There must be no PCR products in the negative 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 additional fainter PCR products evident These additional bands do not interfere with sequence results or quality Page 19 of 25 For In Vitro Diagnostic Use License The SBT Resolver M kits contain GoTaq Hot Start Polymerase DNA POL which 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 Bibliography 1 Sayer D Whidborne R Brestovac B Trimboli Witt C Christiansen 2001 HLA DNA sequencing based typing an approach suitable for high throughput typing including unrelated bone marrow registry donors Tissue Antigens 57 46 54 Sayer D Whidborne R DeSantis D Rozemuller EH Christiansen F Tilanus MG 2004 A multicentre international evaluation of single tube amplification protocols for sequencing based typing of HLA DRBI and HLA DRBS3 4 5 Tissue Antigens 63 412 423 Assign SBT v3 6 Operator Manual Conexio Genomics Pty Ltd Assign SBT v4 7 Operator Manual Conexio Genomics Pty Ltd Assign SBT v471 Operator Manual Conexio Genomics Pty Ltd More information regardin
12. 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 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 thermal parameters cycling Poor PCR purification Ensure ExoSAP treatment is undertaken according to kit s user instructions Ensure that the PCR mixture is mixed thoroughly with ExoSAP Consider using ExoSAP following the manufacturers 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 Page 22 of 25 For In Vitro Diagnostic Use aerosols Contaminated sequencing primer Check sequence quality of the other sequencing primers and other sampl
13. 12 0 0 50 AN PD13 0 0 20 AN PD13 0 0 50 LC PD2 9 20 LC PD2 9 50 Product code CGX0036 Product code CGX00470 Product code CGX00471 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 RB TT197 F 20 RB AT258 F 20 RB GT344 R 20 QB TA185 F 20 PB TAC121 F 20 CI TA98 F 20 C1 GT240 F 20 C1 AT362 F 20 CI CT423 F 20 C1 AC206 F 20 C1 GAT309 R 20 C1 TA420 F 20 C1 AC559 R 20 C1 AG270 F 20 C1 GA361 F 20 RB 15 F 20 RB GT196 F 20 RB GC258 F 20 RB TG344 R 20 QB CG353 R 20 PB GG341 R 20 SBT Resolver HLA DRB4 kit 20 and 50 tests SBT Resolver HLA DRBS 20 and 50 tests SBT Resolver 57 kit 20 and 50 tests Note the products listed above are licensed as IVDs in Australia General Purpose Laboratory Reagents Page 24 of 25 C1 CA102 F 20 C1 TT368 F 20 C1 AC497 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 RB GA196 F 20 RB CT257 R 20 QB GG353 R 20 PB GC194 F 20 For In Vitro Diagnostic Use MgCDP 1 0 50 2mM MgCl2 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 C Cos C
14. ARPS 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 M reference files which are provided by Conexio Genomics Assay Product Code Assign Reference File SBT ResolverTM HLA A XH PDI 1 2 A xml SBT Resolver HLA B BS PD2 1 2 B xml SBT Resolver HLA C HH PD3 2 2 C xml or Cw xml SBT Resolver HLA DRB1 HH PD5 2 5 DRB1 FullX2 xml LG PDS 2 7 527 DRBI xml SBT Resolver HLA DQB1 PQ PD6 2 2 DQB1 xml AN PD6 2 3 623_DQB1 xml SBT Resolver HLA DPB1 HH PD10 1 DPB 1 xml KD PD10 2 1 DPB1 xml Page 17 of 25 For In Vitro Diagnostic Use Performance Characteristics Accuracy Panels of up to 93 samples from the UCLA International DNA Exchange proficiency testing program 2008 2010 used for internal testing for the SBT Resolver kits yielded the following results Locus Number Diagnostic Diagnostic Number Number of Number of sensitivity specificity of heterozygous of samples of of discordant samples unique tested successful genotypes samples alleles PCRs obtained HLA A 81 100 100 0 74 20 HLA B 82 100 98 8 0 79 81 HLA C 39 97 5 97 5 0 35 21 HLA DRBI 93 96 7 96 7 0 84 39 HLA DQBI 42 100 100 0 36 14 PQ PD6 2 2 HLA DQBI
15. DPB1EX1F DPB1EX1R 7 DQB1EX3F DQB1EX3R DPB1EX2F DPB1EX2R TG344 R J amp 3 5 m x 2 N mn DPB1EXSF DPB1EXSR DPB1EX4F DPB1EX4R DPB1EX5F DPB1EX5R PB AG341 R Table 3 Sequencing primers provided for use for each locus RB TG344 R is a HARP directed to the codon 86 dimorphism Its use is optional PB AG341 R is a HARP directed to the codon 85 dimorphism in DPB1 Its use is also optional ADRBIEX3R 2 is sequencing primer in the HH PD5 2 5 kits which behaves similar to a HARP and is designed to sequence the following allele groups 03 08 11 12 13 14 15 and 16 This primer will produce either heterozygous hemizygous or no sequencing data depending on the genotype of the sample being typed When analysing DRBIEX3R 2 data in Assign against the DRBI FullX2 reference the resulting exon 3 data will be analysed in a separate layer and will allow resolution of a number of allele ambiguities in exon 3 such as the DRB1 14 01 vs 14 54 ambiguity Its use is optional depending on the typing strategy used by the laboratory This is not applicable to the LG PD5 2 7 kits as bi directional sequencing for exon 3 is available 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 indicated below are per sample Page 14 of 25 For In Vitro Diagnostic Use Component Volume Se
16. HEROGRAMS ssssssecsssseseessseecsesaeeecsssseceesseseceesaeeeeessaees 17 PERFORMANCE CHARACTERISTICS u ccsssscscssssscccssssscccssssccccesssccccesssscesesssseeceses 18 ACCURACY NEUE UN love ed ee RUE Sous VE CERE PIU ERE AE 18 DETECTION E 5 egere eee o sedate exe deudenedveencstuncedecdovoeee 19 SPECIFICITY e M P RECEN 19 LIMITATIONS AND CAUTIONS eere eee eerte eee tn nee eese seen esee seen eese ee tn esee eoa 19 IB BU kjum m 20 BIBLEIOGRAPHY iere eet ee tesi et costes Peces ve va cuo on DURO evo se ee E en PH Ud see eo cop ve Dono Seen sb UE sadeudes eee 20 TROUBLESHOOTING seriinin c eeee recreo eet ce eere S revo epe e e eee uses este ce o PEDES Ss VoU e PER EUENP 21 RELATED PRODUGCTS e eeeoes ette sees uev edes dose et nee donde seb eP a Sev ue ee uod Res uisa Dee EDO 24 SUPPORT AND CONTACT DETAILS eee e eere ee eerte eee e tone sette ee essen ee eee en ee eese nu 25 Page 2 of 25 For Diagnostic Use Principle The HLA Sequencing Based Typing SBT procedure described here was originally developed by D Sayer in 2001 and developed into a single tube assay in 2004 The procedure involves the initial amplification of the target sequence followed by enzymatic treatment to remove unincorporated primers and dNTPs The amplicon
17. PB1EX3F DPB1EX1R 1x 110uL each DPB1EX2R DPB1EX3R DPB1EX4F DPB1EX4R DPB1EX5F DPB1EX5R PB AG341 R The PRE PCR kit contains a vial of a locus specific PCR mix e g consisting of PCR buffer dNTPs MgCl and locus specific PCR primers along with a single vial of DNA polymerase e g The POST PCR kit contains sequencing primers e g Page 8 of 25 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 the HLA A B C DRB1 DQBI and DPB1 kits indicated a shelf life of two and a half years from 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
18. es using the same primer Consider using a fresh aliquot of sequencing primer Contaminated dye terminator mix or sequencing buffer Repeat sequencing with fresh aliquot of reagents Poor purification of sequencing products Repeat sequencing and ensure that purification is undertaken according to manufacturer s instructions Presence of Dye blobs Poor purification of sequencing products Purify products according to kit instructions Ensure products are washed sufficiently with 8046 ethanol Page 23 of 25 For In Vitro Diagnostic Use Related Products CE marked IVDs ASSIGNz ss ASSIGNssrva7 ASSIGNssrva71 SBT RESOLVER hares Product codes C1 TT98 F 20 C1 CT102 F 20 C1 GG307 R 20 C1 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 RB GG125 F 20 RB TA164 F 20 RB AT257 R 20 QB TA122 F 20 QB TA173 F 20 PB AT251 R 20 PB AG341 R 20 C1 AC98 F 20 C1 CC102 F 20 C1 GG363 AF 20 C1 GT355 R 20 CI BCG F 20 C1 CG319 F 20 C1 GC363 F 20 CI CT559 R 20 C1 GAG601 R 20 C1 CT112 F 20 C1 CG343 F 20 C1 AA601 R 20 RB 04 F 20 RB AA197 F 20 RB TT227 F 20 RB TT321 R 20 QB CT173 F 20 PB GT313 R 20 PB GC112 F 20 For Research Use Only SBT RESOLVER SBT Resolver HLA DRB3 kit 20 and 50 tests AN PD11 0 0 20 AN PD11 0 0 50 AN PD12 0 0 20 AN PD
19. g the UCLA DNA Exchange Program can be found at http www hla ucla edu cellDNA DNA programInfo htm Current HLA alleles can be found at http www ebi ac uk imgt hla Page 20 of 25 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 the mastermix or insufficient mixing of mastermix prior to addition to samples Repeat PCR Ensure mastermix components are 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 before taking gel image
20. les to be tested refer to Table 1 below for the volume per reaction Pulse vortex the solution 3 4 times Locus A B DRB1 DQB1 1 Locus specific PCR Mix 16uL 16 16 16 7uL 16 7uL 16 73 g HLA A MIX DNA Polymerase luL luL luL 0O3gL 03uL 03guL e g DNA POL HLA A 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 positive control s to each reaction well Add 3uL of sterile water to the negative 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 run 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 reaction wells plate from the thermal cycler and either proceed directly to gel electrophoresis or store at 4 C until required NOTE Purification of amplicons by ExoSAP treatment should occur within 24 hours of completion of PCR 2 Agarose Gel Electrophoresis 2 1 Confirm successful amplification by agarose gel electrophoresis using 2uL of each PCR product combined with 5uL loading buffer alternative volumes of loading buffer should be validated prior to use The use of 1 agarose gels is recom
21. mended Page 12 of 25 For In Vitro Diagnostic Use 2 2 The number and expected sizes of the resultant amplicons will vary according to the locus and sample genotype Expected PCR amplicon sizes are indicated in Table 2 Locus Expected band sizes HLA A 2 kbp HLA B 2 kbp HLA C 1 1 kbp and 1 4 kbp HLA DRBI 450bp 850 bp HH PDS 2 5 630 bp 980 bp LG PDS 2 7 Banding pattern will vary depending on the presence of specific allele groups HLA DQB1 300 bp and 500 bp PQ PD6 2 2 400 bp and 500 bp AN PD6 2 3 HLA DPBI 400 bp HH PD10 1 400 bp 780 bp and 1470 bp KD PD10 2 1 Table 2 Expected product sizes for each assay 3 Purification of PCR Product NOTE Purification systems other than ExoSAP IT or ExoProStar e g 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 treatment 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 or ExoProStar and 8uL of 2mM MgCl per sample to be purified Gently pulse vortex to mix Dispense 12uL of the mastermix into the reaction well of each reactive sample Seal the wells vortex and then either place on a shaker or gently vortex for 2 minutes Centrifuge briefly before placing into the thermal cycler Run the thermal cycler according to the followi
22. n wells 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 Page 16 of 25 For In Vitro Diagnostic Use Mobility file KB 3730 POP7 BDTV3 Basecaller KB bcp Run Module Regular FastSeq50_POP7 Injection time 15 sec Run time 3000 sec 6 5 Use the instrument s data collection software to process the raw collected data and create the sequence files Please 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 versions 3 6 and higher as these versions of the software utilise setting and reference files specifically designed for SBT Resolver typing kits and H
23. ncing primers such as SBT Resolver HARPS is recommended 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 samples 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 e The nature of HLA sequence based typing is such that factors other than the PCR mix may result in preferential amplification or allele drop out As a consequence apparent homozygous typing results should be confirmed using alternative methods and or family genotyping positive control human DNA and negative control sterile water must be included on every PCR run The positive control must produce a PCR product of the appropriate size depending on the locus amplified and the
24. ng profile 37 C 30 mins 80 C 15 mins 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 Weaker PCR products may require a lower dilution factor 3 3 ExoS AP treated samples may be stored at 4 C until ready for use These 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 Page 13 of 25 For Diagnostic Use 4 Sequencing Reaction NOTE In instances where heterozygous ambiguities are to be resolved with hemizygous sequencing primers such as HARPS please refer to the SBT Resolver HARPS Instructions for Use 4 Table 3 lists the sequencing primers that are to be used for each locus HLA A HLA B HLA C AEX1F AEX1R BEX1F BEX2F CEX1F CEX1R AEX2F AEX2R BEX3F CEX2F CEX2R AEX3F AEX3R BEX4F CEX3F CEX3R AEX4F AEX4R BEX4R CEX4F CEX4R CEX5F CEX5R CEX6F CEX6R CEX7F HLA DRBI HLA DQBI HLA DPBI DRB1EX2F DRB1EX2R 2 DQB1EX2F DQB1EX2R DPB1EX2F DPB1EX2R DRB1EX3R 24 RB TG344 R DQB1EX3F DQB1EX3R Tt x e DRB1EX2R 2 DRB1EX3R 7 DQB1EX2F DQB1EX2R 3
25. nol with absolute ethanol and sterile water 5 10 Add 60uL of 80 ethanol to each well Reseal the wells and vortex briefly 5 11 Spin at 2000g for 5 minutes 5 12 Repeat steps 5 6 and 5 7 5 13 Remove the reaction wells from the centrifuge and discard the paper towel Reseal the reaction wells 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 6 Denaturation amp Electrophoresis of Sequencing Reaction Products NOTE The procedure for the denaturation of extension products in Hi DiTM 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 well Vortex and centrifuge the wells plate briefly 6 2 Incubate the reaction wells 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 6 3 Load the reactio
26. quencing primer 2uL Sterile water 11 5uL BigDye Terminators luL 5x Seq Rxn Buffer 3 5uL 4 3 Mix each sequencing reaction mixture gently by pulse vortexing 4 4 Dispense 18uL of the sequencing reaction mix into each appropriate reaction well NOTE For runs which involve few samples with many sequencing primers it is acceptable to dispense the sequencing primer 2uL directly into the individual reaction wells A master mix may then be created composing of sterile water BigDye Terminators and 5x Seq Rxn Buffer of which 16uL is to be dispensed into each reaction well It is strongly recommended that use of this alternative procedure is validated by the user prior to implementation 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 wells mix gently and centrifuge briefly to ensure that the contents are located at the base of each reaction well 4 7 Place the reaction wells into a thermal cycler and run according to the following profile Number of cycles Temperature and time 25 96 C 10 sec 50 C 5 sec 60 C 2 min 1 4 C hold 4 8 Once the program is complete remove the reaction wells plate from the thermal cycler and either proceed directly to purification of the reaction products or store in the dark at 4 C until required It is recommended that samples are purified and run on the DNA sequencer within
27. use HLA Sequencing Analysis Software e g Assign SBT version 3 64 or higher Conexio Genomics Pty Ltd Sample Requirements 1 2 Sterile water negative no template control High molecular weight human genomic DNA concentration range of 20 100ng uL in Tris EDTA buffer and OD sooso 1 8 extracted from ACD or EDTA anticoagulated whole blood specimens Do NOT use whole blood specimens containing heparin Page 10 of 25 For In Vitro Diagnostic Use ites 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 patient 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 Tag 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 bet
28. ween 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 under 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 Locus specific PCR Mix a AEX1F HLA A exon 1 forward sequencing primer Refer to Kit Composition and Table 4 for other sequencing primers Date of manufacture required for non EU markets Page 11 of 25 For In Vitro Diagnostic Use Procedure 1 PCR 1 1 A separate PCR reaction will need to be set up for each locus to be amplified and for each individual sample to be tested Each run should include appropriate positive control s of known genotype and at least one negative control for each locus being amplified 1 2 Prepare a fresh solution of PCR master mix each time a PCR is performed Quickly thaw the locus specific PCR mix at room temperature Once thawed vortex briefly 1 3 Dispense the required volume of PCR mix and DNA polymerase into a sterile tube for the number of samp
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