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Protrans Sequencing Testkits (SBT)

1. REF Article 34 01 Protrans 54 HLA A 0197 34 02 Protrans S4 HLA B 60197 34 03 Protrans 54 HLA C 34 04 Protrans S4 HLA DRB1 0197 24 1415 Protrans HLA B 5701 0197 Protrans S3 Allele and Locus Specific Sequencing System REF Article 33 01 Protrans S3 HLA A 60197 33 02 Protrans S3 HLA B 60197 33 03 Protrans S3 HLA C 33 04 Protrans S3 HLA DRB1 0197 33 09 Protrans 53 HLA DQB1 Protrans Haplotype Specific Sequencing System Domino Stones REF Article 54101 13 64101 13 Protrans Domino Stones HLA A 0197 54201 15 54201 15 Protrans Domino Stones HLA B 0197 54301 13 54301 13 Protrans Domino Stones HLA C 544 01 14 64401 14 Protrans Domino Stones HLA DRB1 C 01 97 539 01 09 63901 09 Protrans Domino Stones HLA DQB1 Protrans S2 Allele Group Specific Sequencing REF Article 32 04 Protrans S2 HLA DRB1 60197 32 08 Protrans S2 HLA DQA1 Protrans S1 Locus Specific Sequencing System REF Article 31 01 Protrans 51 HLA A C 01 97 21 01 Protrans S1 HLA A swift 0197 31 02 Protrans 51 HLA B 0197 21 02 Protrans S1 HLA B swift 0197 31 03 Protrans S1 HLA C 31 05 Protrans 61 HLA DRB3 C 0197 31 06 Protrans 51 HLA DRB4 60197 31 07 Protrans 51 HLA DRB5 60197 31 09 Protrans S1 HLA DQB1 Exon 2 3110 Protrans
2. A few minutes before thermocycler program for sequencing reaction ends Fill the columns with Sephadex gel suspension about 34 of the tube 5 Use a plastic Pasteur pipette Centrifuge the Protrans Receiver Tubes with the Filter Columns at Spinning time and g force must be kept exactly 2000x g 90 sec Mark Protrans Receiver Tubes 1 5 ml reaction tubes according to the number of sequencing 8 reactions which have to be purified and place the PROTRANS Filter columns with Sephadex gel suspension in Protrans Receiver Tubes 1 5 ml Purification of Sequencing Products Take one spin column out of Protrans Receiver Tubes 1 5 ml and keep in hand 2 10pl Sequencing Product pipet carefully to the center of the angled surface of the pre spinned Sephadex column Pay attention that the tip is not touching the Sephadex gel Careful application of the sample to the center of the bed is essential for good separation Do not allow any of the sample to flow around the sides of the Sephadex column Place spin filter back in marked 1 5ml tube Spin Protrans column down at exactly 2000xg 1 Min Spinning time and g force must be kept exactly Until loading the marked sequencing products should be stored at 4 86 or for longer periods at 20 protected from light Sequencing set up 1 18ul HPLC water H pipet in a MicroAmp Optical plate 2 2yl Purified Sequencing Product p
3. PROTRANS AmpliPUR Fast for purification of PCR products 1 EXoSAP IT Exonuclease l Shrimp Alkaline Phosphatase for purification of PCR products MicroAmp optical 96 well reaction Plate Part Number N801 0560 Automated DNA sequencing instrument and consumables ABI Sequencer 1 Big Dye Terminator Cycle Sequencing Kit version 1 1 v 1 1 2 CEQ 8000 QuickStart Kit Beckmann Coulter 1 MegaBACE DYEnamic ET Dye Terminator Kit MegaBACE PROTRANS PROTRANS PROTRANS multiple suppliers multiple suppliers multiple suppliers multiple suppliers multiple suppliers PROTRANS multiple suppliers multiple suppliers multiple suppliers PROTRANS USB Europe GmbH www usbweb de Art Nr 78201 78202 ABI Applied Biosystems Beckman Coulter GE Healthcare Applied Biosystems Beckman Coulter GE Healthcare 20 Protrans User Manual SBT Version 2010 07 01 Note 1 Applied Biosystems The Protrans HLA Sequencing Kits have been validated for the Applied 2 Beckman Coulter Biosystems DNA Sequencers 3100 Genetic Analyzer and 3730 DNA Analyzer SS They should also work with all other four dye sequencing instruments available MegaBACE GE Healthcare PROTRANS DyePUR for purification of sequencing products PROTRANS Sephadex G 50 Fine DNA Grade for purification of sequencing products GE Healthcare 96er plate centrifuge for purification of sequencing products on 96 well plates Ethanol or
4. 2 3 and 4 to Sort out nearly all ambiguities caused by variations outside Exons 2 and 3 Special emphasis was put on the location of the sequencing primers to ensure complete Exon sequences in both orientations For ease of use The Group Specific Primer Mixes are pre pipetted in 8 well PCR Strips e Test procedure and the Thermocycler program for the Amplification Cycle Sequencing and the Settings of the Sequencer are for all Protrans Sequencing Kits and for all HLA loci identical e The Purification of the PCR Products and Sequencing Products are identically for all HLA loci e It is very easy to type different HLA loci of several DNA Samples in parallel e Itis always possible to combine the different Protrans Sequencing Kits in the different Protrans Sequencing Strategies Protrans Domino Stones HLA SBT are designed for a maximal flexibility to match individual requirements of the HLA laboratory The Domino Stones Locus and different Group Specific PCR Amplification Mixes Domino Stones are supplied separately to allow an individual set up according to the individual requirements Using the Protrans Domino Stones it is for all HLA loci possible to change the low resolution typing result from other techniques SSP or SSO in a 4 digit high resolution result The Locus and Group Specific PCR Amplifications are covering at least Exons 2 and 3 in HLA class loci and Exon 2 in HLA class II loci Protrans User Manual SBT
5. 4 86 16 Protrans User Manual SBT Version 2010 07 01 Testkit Tubes Sequencing i i I Primers Exon 2 DPB 1 2 forward blue E2F x 49 06 1x 360 20 Agarose Gel Loading Buffer colourless LB 1x 400 4 86 Content of the Sequencing Unit components Sequencing Primers 5 and 3 primers Loading Buffer Gel Loading Solution Note Refer to the Primer Mix Specificity Tables Attachment delivered with the kits to select the appropriate PCR products and Sequencing Primers Make sure that the version of the kit and the version of the Primer Mix Specificity Tables are identical Note All reagents supplied with the Protrans HLA Sequencing Kits should be used Amplification Primer Version specific and Sequencing Primer LOT specific Amplification Primer can not be mixed between different Versions as indicated on the Attachment of each Kit Sequencing Primer can not be mixed between different LOT s as indicated as indicated on the Attachment of each Kit Note In case of damaged tubes or boxes malfunctions cannot be excluded Those Kits must not be used Protrans User Manual SBT Version 2010 07 01 17 5 0 Storage and Shelf Life Pre PCR Area 4 Amplification Unit 1 Precoated Strips 4 8 Amplification Unit 2 Buffers 20 Sequencing Unit Sequencing Primers 20 When stored under appropriate conditions the kit comp
6. Protrans S3 HLA Sequencing Kit negative control should be performed for each series of DNAs tested to exclude contamination of the PCR solution and AmpliTaq Gold DNA Polymerase Protrans User Manual SBT Version 2010 07 01 27 10 6 PROTRANS Domino Stones HLA SBT Sequencing Kits The Protrans Domino Stones are designed for a maximal flexibility to match individual requirements of the HLA laboratory The Domino Stones Locus and different Group Specific PCR Amplification Mixes Domino Stones are supplied separately to allow an individual set up according to the individual requirements Using the Protrans Domino Stones it is for all HLA loci possible to change the low resolution typing result from other techniques SSP or SSO in a 4 digit high resolution result The Locus and Group Specific PCR Amplifications are covering at least Exons 2 and 3 in HLA class loci and Exon 2 in HLA class II loci For ease of use e The Test procedure and the Thermocycler program for the Amplification Cycle Sequencing and the Settings of the Sequencer are for all Protrans Sequencing Kits and for all HLA loci identical e The Purification of the PCR Products and Sequencing Products are identically for all HLA loci e It is very easy to type different HLA loci of several DNA Samples in parallel e Itis always possible to combine the different Protrans Sequencing Kits in the different Protrans Sequencing Strategies 10 7 Set up Amplification Pr
7. for detailed information Available from Protrans 18 Protrans User Manual SBT Version 2010 07 01 6 1 Materials and Equipment not supplied with the Kit SEQUENCE PILOT Software for automated allele assignment PROTRANS PROTRANS Pipetting Assistant PPA Software for documentation of DNA samples pipetting regime PROTRANS Transfer of generated information to the Platerecord of the sequencer DNA Extraction PROTRANS DNA Box 500 PROTRANS Photometer for adjusting DNA concentration multiple suppliers Protrans PCR Workstation deep frozen block for pipetting PCR PROTRANS Vortexer multiple suppliers Mini Centrifuge multiple suppliers AmpliTaq Gold polymerase 5U ul Applied Biosystems Eppendorf Tubes 1 5ml Rainin L 20 Pipet Lite PROTRANS Rainin L 1000 Pipet Lite PROTRANS Pipettes filter tips PROTRANS Closing roller PCR caps multiple suppliers Thermal cycler with heated lid Note The PCR and sequencing cycle profiles provided in this manual have been used with the Thermal Cyclers from Applied Biosystems GeneAmp PCR multiple suppliers Systems 2700 9600 and 9700 They should also work with compatible instruments but may require adjustments of the cycling profile or emulation of the above instruments See Appendix A troubleshooting guide Protrans User Manual SBT Version 2010 07 01 19 6 2 Materials and Equipment not supplied with the Kit Rainin L 20 Pipet Lite Rai
8. x x x x X A E3R 1x 5x 360 20C reverse blue Exons x x x A E4F 1x 360 20 forward yellow EXOR 4 x x x A E4R 1x 360 20 reverse yellow Agarose Gel Loading Buffer colourless LB 1x S4 2x 400 4 80 Testkit Tubes i Do Sequencing s s3 s1 stsw P 54 53 82 Do st ul 4 Primers St exon x x x B E1F 1x 360 206 forward natural Exon x x x B E1R 1x 360 206 reverse natural 1x 5x 360 20C 1x 5x 360 206 Exon 3 x x x x x B E3F 1x 5x 360 20 forward blue En x x x x x B E3R 1x 5x 360 20 reverse blue EXON x x x B E4F 1x 360 20 forward yellow B E4R 1x 360 20 reverse yellow Agarose Gel Loading Buffer colourless LB 1 542 400 4 80 14 Protrans User Manual SBT Version 2010 07 01 Protrans HLA C Testkit Tubes Sequencing c s3 gi Do tube 54 3 52 P gt d St St Exon 1 forward natural in preparation C E1F Exon 1 r veres natural in preparation C E1R 1x 5x 360 206 1x 5x 360 206 1x 5x 360 206 1x 5x 360 206 EXER x x C E4F 1x 360 20 forward yellow rond x x C E4R 1x 360 2
9. 53 HLA DQB1 Specificity Group 1 DQB1 0301 0304 0309 0310 0312 0313 DQ3 Group 2 DQB1 0302 0303 0305 03080 0311 fers oem eese Fees EE B 07 48 8101 B 4802 B 08 42 B 15 45 46 49 50 4026 4427 B 18 37 B 27 3542 4002 group 47 82 w o B 270504 B 35 4802 51 52 53 5606 58 78 B 14 38 39 6701 B 4001 group 41 B 42 w o B 4202 B 44 8301 w o B 4415 4418 B 54 55 56 5901 w o B 5606 B 57 All HLA B Alleles Neg Amplification Control 54 HLA DRB1 Specificity DRB1 01 DRB1 15 16 DRB1 03 1315 1402 1406 1413 w o DRB1 0317 1417 DRB1 04 DRB1 03 11 13 14 0806 w o DRB1 1317 DRB1 08 11 13 14 w o DRB1 1402 1406 1413 DRB1 03 11 13 1402 1403 1406 1413 1417 1421 DRB1 0806 w o DRB1 0317 1313 DRB1 12 DRB1 1301 1302 1334 1417 1421 DRB1 14 1117 w o DRB1 1402 1403 1406 1413 1417 DRB1 07 DRB1 08 1317 DRB1 0806 DRB1 09 DRB1 10 Positive Amplification Control Negative Amplifikation Control Foto Protrans User Manual SBT Version 2010 07 01 14 0 Purification of the Haplotypes Purification of the positive PCR Products The PCR products have to be purified before using them as sequencing templates because residual PCR primers and nucleotide triphosphates dNTPs can inter
10. Amplification Protrans S4 Sequencing Kits Documentation of DNA samples with Protrans Pipetting Assistant or pipetting scheme form Place reagents and strips in PROTRANS PCR Workstation 20 1 16 well PCR Strip 2 PROTRANS PCR Solution D PSD 3 AmpliTaq Gold DNA Polymerase 4 DNA sample Vortex all reagents and spin down with mini table centrifuge Reaction tube 1 5ml Master Mix for each DNA sample 1 280 ul PCR Solution PSD 2 3 0 pl AmpliTaq Gold DNA polymerase Vortex master mix and spin down with mini table centrifuge 3 15ul Master Mix in Position 14 Negative Control N Protrans 16 well PCR Strip 4 20ul DNA sample 50 100ng ul in the Master Mix Vortex master mix and spin down with mini table centrifuge Master Mix in Protrans 16 well PCR Strip 134 with electronic Multistepper RAININ Close 16 PCR strip Place the strips in the thermocycler and start the PCR amplification program Protrans User Manual SBT Version 2010 07 01 25 10 3 Protrans S3 Sequencing Kits 26 The Protrans S3 Sequencing Kits are designed to match the requirements of high sample throughput as well as allele specific sequencing for less ambiguities This is achieved by splitting the Haplotypes before sequencing each Haplotype separately In the Protrans SBT Test kit HLA A B C the DNA will be amplified with 7 Group Specific PCR Amplification
11. DNA Evaluate on agarose gel and re extract the DNA if necessary DNA not measured Re measure DNA and adjust to 50 100 correctly ng ul PCR inhibitors in the Re extract genomic DNA using one of the genomic DNA recommended methods see Preparing Sample DNA avoid using blood treated with heparin Inappropriate Check the cycling profile and correct it if cycling parameters necessary Thermal cycler The cycling profiles provided in this manual problems are optimized for the GeneAmp PCR Systems 2400 2700 9600 and 9700 Applied Biosystems as well as the PTC 100 PTC 200 Dyad and Tetrad MJ Research The use of other thermal cyclers may require adjustment of the cycling profile 2 PCR bands Inappropriate Run agarose gel according to appropriate agarose gel conditions conditions Non specific Inappropriate Check the cycling profile and correct it if PCR bands cycling parameters necessary Thermal cycler problems The cycling profiles provided in this manual are optimized for the GeneAmp PCR Systems 2400 2700 9600 and 9700 Applied Biosystems as well as the PTC 100 PTC 200 Dyad and Tetrad MJ Research The use of other thermal cyclers may require adjustment of the cycling profile Inappropriate DNA Taq polymerase Repeat PCR with AmpliTag Gold DNA polymerase Contamination of the PCR reagents Check the related PCR negative control to exclude contamination of the PCR reagents Use filter tip
12. Exon 2 Exon 3 and Exon 4 The Single Allele or Group Specific Primer Mixes are pre pipetted in 16 well PCR Strips In almost all cases sequence analysis of separately both alleles will be achieved In HLA class special emphasis was put on the complete coverage of Exons 1 2 3 and 4 to sort out nearly all ambiguities caused by variations outside Exons 2 and 3 In HLA class Il the DNA will be amplified with up to 14 Group Specific PCR Amplifications GSA parallel covering the complete Exon 2 loci The Single Allele or Group Specific Primer Mixes are pre pipetted in 16 well PCR Strips In almost all cases sequence analysis of both alleles separately will be achieved In HLA DRB1 a special emphasis was put on the separation of the DR52 associated HLA DRB1 alleles to ensure unambiguous results in nearly all samples Special emphasis was put on the location of the sequencing primers to ensure complete Exon sequences in both orientations For ease of use The Group Specific Primer Mixes are pre pipetted in 8 well PCR Strips e Test procedure and the Thermocycler program for the Amplification Cycle Sequencing and the Settings of the Sequencer are for all Protrans Sequencing Kits and for all HLA loci identical Purification of the PCR Products and Sequencing Products are identically for all HLA loci e tis very easy to type different HLA loci of several DNA Samples in parallel e Itis always possible to combine the d
13. GSA 24 4 8 Protrans S2 HLA DQA1 Pri Mi Allele Group specific Sequencing Typings 4 well PCR Strips precoated with Primer Mixes 4x GSA 24 4 8 Protrans S3 HLA DQB1 I Single Allele and Locus Specific Sequencing 8 well PCR Strips pink precoated with Primer Mixes 6x GSA 2xLSA 24 4 86 Protrans User Manual SBT Version 2010 07 01 9 PCR Solution D PSD 1 750 20 PCR Solution D PSD 1 750 20 Negative control NC ABC 1x 140 20 Allele and Allele Group specific Amplification GSA 1x 1 400 20 PCR Solution L 3x 1 000 20 Locus specific Amplification Primer LSA 360 20 Locus specific Amplification Primer LSA 360 20 10 Protrans User Manual SBT Version 2010 07 01 PCR Solution D PSD 4x 1 750 206 PCR Solution D PSD 2x 1 750 20 Negative control NC ABC 1x 140 20C Allele and Allele Group specific Amplification GSA 1x 1 400 200 PCR Solution L PSL 3x 1 000 20 Locus specific Amplification Primer LSA 1x 360 206 Locus specific Amplification Primer LSA 1x 360 206 Amplification Unit 2 s pis Solution Tubes Volume 4 Single Allele and Locus specific Sequencing PCR Solution D PSD 4x 1 750 200 Protrans S3 HLA C I Sing
14. Version 2010 07 01 5 Protrans S2 Sequencing Kits The Protrans S2 sequencing kits are designed to match the requirements of very high sample throughput as well as allele specific sequencing for less ambiguities This is achieved by applying 4 Group Specific PCR Amplifications GSA in parallel covering at least exons 2 and 3 in HLA class loci and exon 2 in HLA class II loci allowing in many cases sequence analysis of both alleles separately For ease of use The Group Specific Primer Mixes are pre pipetted in 8 well PCR Strips e The Test procedure and the Thermocycler program for the Amplification Cycle Sequencing and the Settings of the Sequencer are for all Protrans Sequencing Kits and for all HLA loci identical e Purification of the PCR Products and Sequencing Products are identically for all HLA loci e It is very easy to type different HLA loci of several DNA Samples in parallel e Itis always possible to combine the different Protrans Sequencing Kits in the different Protrans Sequencing Strategies Protrans S1 Sequencing Kits The Protrans S1 sequencing kits are designed to match the requirements of very high sample throughput This is achieved by Locus Specific PCR Amplification LSA covering in HLA class Exon 1 Exon 2 Exon 3 and Exon 4 and in HLA class Exon 2 allowing sequence analysis of both alleles simultaneously A special emphasis was put on the location of the sequencing primers to ensure comple
15. a signal range from 100 2000 relative fluorescent units The finally prepared Increase dilution in the final step of sequencing reaction preparing sequencing reactions for was too electrophoresis 36 ul instead of 18 ul concentrated HPLC water Noisy Inappropriate PCR See Purifying PCR products for baseline product purification Sequencing or purification omitted before sequencing Inappropriate sequencing reaction purification Re purify the sequencing reaction see Purifying Sequencing Reactions Remember to pipet the complete sequencing reaction carefully on the center of the sloping Sephadex surface without touching it Pipetting on the center is essential for high quality purification Do not allow the sample to flow beside the resin User Manual SBT Version 2010 07 01 45 Sequencing Trouble shooting Table continued Noisy Contamination of Check the related PCR negative control to baseline the PCR product exclude contamination of the PCR reagents To exclude contamination of the DNA sample re extract DNA from a new source sample Use filter tips Contamination of Repeat sequencing with a fresh tube of the sequencing sequencing primer Use filter tips primer Contamination of Repeat sequencing with a fresh tube of dye the dye terminator terminators Use filter tips reagents Broad Inappropriate Re purify the sequencing reaction fluorescent sequencing
16. primers with mini table centrifuge 6 6ul Sequencing Primers dispense to the left side of the wells in the H MicroAmp optical Plate RAININ Multistepper 7 2yl Purified PCR Product pipet in the middle of the wells in the MicroAmp optical plate RAININ Multistepper Seal the plate carefully and place the plate in the thermocycler Start the cycle sequencing program Note If sequencing reactions give to strong signals a dilution of the RR Mix might be appropriate beginning with a 1 2 dilution Dilute Big Dye Terminators 1 1 with 5x Sequencing Buffer 1 2 Note If sequencing reactions give rise to background signals or a to strong signal decline a dilution of the PCR product may be appropriate beginning with a 1 2 dilution Protrans User Manual SBT Version 2010 07 01 37 17 0 Purifying Sequencing Reactions 17 1 PROTRANS DyePUR 38 The sequencing products have to be purified to remove unincorporated dye terminators The PROTRANS DyePUR purification kit for purifying sequencing reactions is based on spin filtration using Sephadex G 50 and PROTRANS DyePUR columns The method provides high quality sequence data Preparation of the Sephadex gel for 20 columns 50 Falcon tube 2 1g Sephadex G 50 DNA Grade F 3 12ml HPLC Water Merck 4 Incubation at RT mix or vortex several time during incubation 30 min Mark the Protrans Receiver Tubes 2ml
17. reaction Remember to pipet the complete terminator purification sequencing reaction carefully on the center artifacts dye blobs of the sloping Sephadex surface without touching it Pipetting on the center is essential for high quality purification Do not allow the sample to flow beside the resin High fluorescent artifact peaks Air bubbles in the capillary or polyacrylamide gel Capillary DNA sequencer Refill the capillaries Consider possibly to change the capillary array or to contact the manufacturer s service for your sequencing instrument Slab gel DNA sequencer Pour the gels carefully and appropriately treat the gel sides of the glass plates to avoid air bubbles Contact the manufacturer s service for your sequencing instrument Protrans medizinische diagnostische Produkte GmbH Ketschau 2 68766 Hockenheim Germany Telephone 4449 6205 29299 0 Telefax www protrans info mail protrans info 46 Protrans 49 6205 29299 20 O OT On Ss medizinische diagnostische produkte gmbh User Manual SBT Version 2010 07 01
18. the laboratory s individual requirements Sequencing a gene will give the most reliable and accurate information of the DNA The quantity of known and permanently new identified HLA genes in the human major histocompatibility complex MHC requires a high resolution unambiguous and precise typing method for HLA Alleles Class and Class ll The Protrans Sequencing System is a practical reliable and adjustable typing method With the Protrans Sequencing System it is easy to type all HLA loci in the daily routine low or high throughput formats manually or at each level of automation and resolution desired Protrans Sequencing System is easy to use not only for application in the bone marrow registry but also as diagnostic tool for patient characterization in daily routine With the Software SEQUENCE PILOT it is easy to do the analysis of the Sequencing Files of the Protrans Sequencing System for HLA Class und Class Il Protrans Sequencing Strategies Splitting the Haplotypes 1 PROTRANS S4 single allele and Locus specific amplification and sequencing No Ambiguities Splitting the Haplotypes 2 PROTRANS S3 single allele and locus specific amplification and sequencing No Ambiguities PROTRANS Haplotype specific sequencing No Ambiguities Domino Stones Domino Stones get your LCT SSP SSO results high Splitting the Haplotypes 4 PROTRANS S2 HLA group specific amplification and sequencing 5 PROTRANS S1 HLA locu
19. 0 reverse yellow Agarose Gel Loading Buffer colourless LB 1 542 400 4 8 Protrans HLA DRB1 Testkit Tubes Sequencing c s3 s2 Do St Tube 94 53 52 Primers St 1x 5x 360 206 1x 5x 360 200 DR Exon 2 x x x x codon 1x 1 360 20 Codon 86TG 86TG Agarose Gel Loading Buffer colourless LB 1 542 400 4 86 Protrans HLA DRB 3 4 5 Testkit Tubes sequencing S1 Tube S1 ul Primers 1x 360 206 1 360 206 Agarose Gel Loading Buffer colourless LB 1x 400 4 8 Protrans User Manual SBT Version 2010 07 01 15 Protrans HLA DQB1 Exon 2 Testkit Tubes Sequencing Do Primers S1 Do St Tube S1 St ul 1 5x 360 20C 1x 5x 360 20C Agarose Gel Loading Buffer colourless LB 1x 400 4 8 Protrans HLA DQB1 Exon 3 Testkit Tubes sequencing S1 Do St Tube S1 De Primers St 1x 5x 360 20C 1x 5x 360 206 Agarose Gel Loading Buffer colourless LB 1x 400 4 80 Protrans HLA DQB1 Exon 2 3 Testkit Tubes S3 Do St Tube S3 Primers St 2x 5x 360 20C 2x 5x 360 20C Agarose Gel Loading Buffer colourless LB 1x 400 4 80 Protrans HLA DQ A1 Testkit Tubes SEHR S2 Tube ER ul Primers 1x 360 206 1x 360 206 Agarose Gel Loading Buffer colourless LB 1x 400
20. 1 31 32 2 PCR are positive 2 Haplotypes If two PCRs are positive both PCR products must be sequenced each in a single orientation forward or reverse If more than two PCRs are positive those PCR products with the least specificity overlap must be sequenced each in a single orientation forward or reverse If the PCR amplification pattern is not consistent with the Primer Mix Specification Tables e g by indicating the presence of more than two alleles a contamination or a variant allele may exist If the negative control excludes contamination single orientation sequencing of the PCR products with the least specificity overlap must be performed If the sequencing results give identical sequences of these PCR products the other Group Specific PCR products have to be sequenced to exclude homozygosity Only one PCR is positive If only one PCR is positive the LSA PCR product or in HLA DRB1 this GSA PCR product must be sequenced in both orientations forward and reverse Note The HLA DRB1 sequencing kits are based on group specific sequence diversities outside exon 2 For several HLA DRB1 alleles these sequences are not available yet It has been shown that outside exon 2 HLA DRB1 alleles are conserved within allelic groups and are thus amplified by the dedicated primer mix However it cannot be completely excluded that some alleles with unknown motifs outside exon 2 are not covered by the HLA DRB1 primer mixes It is important to b
21. CR Solution D PSD 2x 1 750 20 Negative control NC DQ 1x 140 20 Allele and Allele Group specific Amplification GSA 1x 1 400 20C PCR Solution L PSL 3x 1 000 20 Locus specific Amplification LSA 1x 360 20 Locus specific Amplification LSA 1x 360 20 12 Protrans User Manual SBT Version 2010 07 01 PCR Solution D PSD 1 1 750 20 Locus specific Amplification LSA 1x 360 20 Content of the Amplification Unit components 5 and 3 primers Protrans S4 precoated Strips Cresol red in the negative control well last position Protrans S3 precoated Strips 5 and 3 primers Protrans S2 precoated Strips 5 and 3 primers Protrans Domino Stone Primer Mix 5 and 3 primers 5 and 3 primers Protrans S1 LSA Mix Buffer Nucleotides Buffer PCR Solution D PSD Nucleotides Buffer PCR Solution L PSL Nucleotides Negative Control NC 5 and 3 primers Protrans User Manual SBT Version 2010 07 01 13 Testkit Tubes i Do Do Sequencing s s3 s1 stsw P gt Tube 54 53 82 P gt d Primers St St Xon x x x A E1F 1x 360 20 forward natural Exon x x x A E1R 1x 360 20 reverse natural 1x 5x 360 206 1x 5x 360 200 Exon 3 x x x A E3F 1x 5x 360 20 forward blue Pron
22. D 2 0 75 pl AmpliTaq Gold DNA polymerase Vortex master mix and spin down with mini table centrifuge 3 5 ul DNA sample 50 100ng ul in the Master Mix Vortex master mix and spin down with mini table centrifuge Master Mix in Protrans 8 well PCR Strip with electronic Multistepper RAININ 4 15u Close 16 PCR strip Place the strips in the thermocycler and start the PCR amplification program Protrans S2 HLA Sequencing Kit negative control should be performed for each series of DNAs tested to exclude contamination of the PCR solution and AmpliTaq Gold DNA Polymerase Protrans User Manual SBT Version 2010 07 01 29 10 11 Protrans S1 Sequencing Kits The Protrans S1 Sequencing Kits are designed to match the requirements of very high sample throughput as well as extended sequencing in HLA class to sort out all ambiguities caused by variations outside exons 2 and 3 This is achieved by Locus Specific PCR Amplification LSA covering exons 1 2 3 and 4 in HLA class Loci and exon 2 in HLA class 11 loci allowing sequence analysis of both alleles simultaneously A special emphasis was put on the location of the sequencing primers to ensure complete exon sequences in both orientations For ease of use the Locus Specific Amplification Mix is ready for use A variant of the Protrans S1 kit design is the Protrans S1 swift Sequencing Kit The Protrans S1swir Sequencing Kit is designed to ma
23. E2R Codon 86TG In most cases sequencing of exons 2 and 3 may be sufficient even for 4 digit typing and is always sufficient if only a 2 digit typing information is required The sequences of exons 1 and 4 may be required to sort out certain ambiguities as well as non expressed null alleles Attachment see kit insert 36 shows Specificities of the Amplification Primer and additional possibilities to use Sequencing Primers for Exon 1 or Exon 4 Exon 1 reverse sequencing may also be possible by extending the sequencing run of Exon 2 reverse Exon 2 forward sequencing may also be possible by extending the sequencing run of Exon 1 forward However this requires longer high quality reads and specific settings of the sequencing instrument Moreover in certain allele combinations a deletion in intron 1 causes a chromatogram shift inhibiting sequence analysis of Exons 1 and 2 respectively Protrans User Manual SBT Version 2010 07 01 16 0 Instruction Set up Sequencing reaction 3 dot Method 1 Generate sequencing plate with Protrans Pipetting Assistant Protrans SEQ 1 or Pipetting Scheme form PCR plate or MicroAmp optical plate In Protrans Bia DveT inat PCR Workstation 200 2 9 Ye terminators Sequencing primers 3 Vortex Big DyeTerminators Big Dyes dispense to the left side of the wells in the 4 2ul MicroAmp optical plate RAININ Multistepper 5 Spin down sequencing
24. Mixes GSA and in addition a Locus specific Amplification Mix LSA In the Protrans SBT Test kit HLA DQB1 the DNA will be amplified with 6 Group Specific PCR Amplifications Mixes GSA and in addition 2 Locus specific Amplification Mixes LSA In the Protrans SBT Test kit HLA DRB1 the DNA will be amplified with 8 Group Specific PCR Amplifications GSA If the GSA reactions do not indicate two separate alleles the LSA reaction must be sequenced This ensures in all cases the recognition of both alleles In the Protrans SBT Test kit HLA class Exons 1 Exon 2 Exon 3 and Exon 4 and in the Protrans SBT Test kit HLA class Exon 2 DRB1 DQB1 and Exon DQB1 are covered The Amplification Primer Mixes are pre pipetted in 8 well PCR Strips and each HLA locus in a different color In most cases sequence analysis of separately both alleles will be achieved In HLA class special emphasis was put on the complete coverage of Exons 1 2 3 and 4 to Sort out nearly all ambiguities caused by variations outside Exons 2 and 3 Special emphasis was put on the location of the sequencing primers to ensure complete Exon sequences in both orientations For ease of use The Group Specific Primer Mixes are pre pipetted in 8 well PCR Strips e Test procedure and the Thermocyclerprogram for the Amplification Cycle Sequencing and the Settings of the Sequencer are for all Protrans Sequencing Kits and for all HLA loci identical e Pu
25. PROTRANS PlatePUR DREES EDTA di Sodium 0 5M Merck and other Na Acetate 3M solution Merck and other Ethanol absolute pro analysis 100 for ethanol precipitation method Merck HPLC water Merck Aluminium cover foil for ethanol purification PROTRANS 7 0 7 1 Note The reagents and instruments listed with 1 have been validated for Protrans HLA Sequencing Kits Protrans does not take any responsibility when other materials or equipment are used The user must validate reagent and instruments other than those listed with Note The reagents and instruments listed with 2 have not been validated for Protrans HLA Sequencing Kits Protrans does not take any responsibility when these materials or equipment are used The Reagents and instruments listed with must be validated by the user Preparation and Processing of Samples DNA Extraction and DNA concentration Genomic DNA can be obtained from all nucleated cells The simplest method is to isolate DNA from cell suspensions blood buffy coat or cultured cells for this multiple protocols and kits are available For DNA sequencing only those methods should be considered which provide DNA of high quality and quantity PROTRANS DNA Box 500 The PROTRANS DNA extraction kit PROTRANS DNA Box 500 provides high quality DNA optimized for PROTRANS HLA sequencing kits Other extraction methods must be validated before used in routine sequencing Note Use either EDTA or ACD blood Heparin anticoagulated bl
26. S1 HLA DQB1 Exon 3 31 11 Protrans 51 HLA DPB1 Protrans S4 24 Protrans S3 24 Protrans 52 24 Protrans S1 24 24 Protrans Domino Stones 240 IVD For In Vitro Diagnostic use Amplification Unit 1 pre pipetted PCR Strips 4 8T Amplification Unit 2 20 Sequencing Unit 200 2 Protrans User Manual SBT Version 2010 07 01 1 0 PROTRANS Sequencing Strategy The Protrans Kits should be used in accordance with the current guidelines for quality assurance established by the European Federation for Immunogenetics EFI or the American Society for Histocompatibility and Immunogenetics Ashi Sequencing gives the most reliable and accurate information of the DNA sequence of a gene and is therefore of particular interest to fully characterize the genetic complexity and allelic diversity of the HLA genes in the human major histocompatibility complex MHC The full complexity of allelic diversity in HLA class and class makes PCR SBT the method of choice for HLA typing HLA typing by means of sequencing should be considered whenever the HLA type of an individual is needed The recent developments have made sequencing equally simple and robust making it attractive for patient related diagnostic and bone marrow registry typing With the Protrans Sequencing System it is easy to type in low or high throughput formats at each level of automation and resolution desired Sequencing can be carried out manually or fully automated depending on
27. Sequencing Products on all Sequencers Automated Analysis of the Sequencing results Sequence Pilot Protrans Software 8 Protrans User Manual SBT Version 2010 07 01 4 0 Materials supplied with the HLA Sequencing Kits 16 well PCR Strips yellow precoated with Primer Mixes 8 well PCR Strips yellow precoated with Primer Mixes 1x LSA 1x neg ctrl 16 well PCR Strips blue precoated with Primer Mixes 8 well PCR Strips blue precoated with Primer Mixes 12x GSA 1x neg ctrl 24 24 4 86 Protrans S4 HLA C Single Allele and Locus specific Sequencing up BEC Typings i 16 well PCR Strips green precoated with Primer Mixes 1 es 24 4 8 GSA LSA neg ctrl Protrans S3 HLA C I Single Allele and Locus specific Sequencing 8 well PCR Strips green precoated with Primer Mixes 7x GSA 1xLSA 24 4 86 Protrans 54 HLA DRB1 Pri Mi Single Allele and Allele Group specific Sequencing Typings 14x 1x 1x 16 well PCR Strips natural precoated with Primer Mixes GSA pos ctrl neg ctrl 24 4 8 Protrans S3 HLA DRB1 I Single Allele and Allele Group specific Sequencing 8 well PCR Strips natural precoated with Primer Mixes 8x GSA 24 4 86 Protrans S2 HLA DRB1 Pri Mi Allele Group specific Sequencing Bienes Typings 4 well PCR Strips natural precoated with Primer Mixes 4x
28. User Manual PMOTMNS HLA SBT O OT O YS medizinische diagnostische produkte gmbh Protrans medizinische diagnostische Produkte GmbH D 68766 Hockenheim Ketschau 2 Germany ul Tel 49 0 6205 29299 0 Fax 49 0 6205 29299 20 www protrans info mail protrans info Contents Page Protrans HLA SBT Testkits 1 1 0 Protrans Sequencing Strategy 3 2 0 PROTRANS HLA Sequencing System 4 20 PROTRANS Sequencing Kits S4 53 Domino Stones 52 51 4 3 0 Protrans Sequencing Procedure 8 4 0 Materials supplied with the HLA Sequencing Kits 9 5 0 Storage and Shelf Life of Testreagents 17 6 0 Precautions and Warnings 17 6 1 Materials and Equipment not supplied with the Kit PRE PCR 18 6 2 Materials and Equipment not supplied with the Kit POST PCR 19 7 0 Preparation and Processing of Samples 20 7 1 DNA Extraction and DNA concentration 20 8 0 Program Thermocycler Protrans SBT 21 9 0 Specificities of the Amplification Primers 22 10 0 PROTRANS S4 Sequencing Kits 22 10 1 PROTRANS S4 Sequencing Kits pre coated PCR Strips 23 10 2 Set up Amplification PROTRANS S4 Sequencing Kits 24 10 3 PROTRANS S3 Sequencing Kits 25 10 4 PROTRANS S3 Sequencing Kits pre coated PCR Strips 26 10 5 Set up Amplification PROTRANS S3 Sequencing Kits 26 10 6 PROTRANS Domino Stones Sequencing Kits 27 10 7 Set up Amplifica
29. certain rules of sample naming conventions must be followed Even if you are not using the automated joining features of the Sequence Pilot software sample naming conventions should be followed to simplify clarity for manual processes Enter the following types of information in brackets SamplelD Amplification Mix Sequencing Primer any other information Alleles Separated The sequencing reactions of a DNA sample that was amplified by the Group Specific Mixes S4R2 DRB1 15 16 and S4R4 DRB1 04 have the following file names Example SamplelD S4R2 DR E2F SamplelD S4R4 DR E2F corresponds to Haplotype 1 forward corresponds to Haplotype 2 forward Alleles not separated The sequencing reactions of a DNA sample that was amplified by the single Group Specific Mix S4R2 DRB1 15 16 have the following file names Example SamplelD S4R2 DR E2F SamplelD S4R2 DR E2R corresponds to Haplotype 1 2 forward corresponds to Haplotype 1 2 reverse Due to these sample naming conventions the Sequence Pilot Software automatically recognizes that these sequencing results belong together and performs allele assignment based on both result files Protrans User Manual SBT Version 2010 07 01 22 0 Performing Allele Identification Result Files Me nu Mark the result file s belonging to the same Haplotype s of the DNA sample Select save 1 to transfer them as joined or paired result files in the joined re
30. channel multi dispensing pipette Close the plate 17 1 Incubate at 95 C Thermocycler 2 17 2 Keep on Protrans PCR Workstation 2 min Protrans User Manual SBT Version 2010 07 01 39 17 3 Alternative Purification methods comprise amongst others Agencourt CleanSEQ beads purification technology Ethanol precipitation or Millipore Montage filter technology which have all been shown to yield high quality data These techniques can also be applied in a 96 well format for high throughput requirements Moreover the Agencourt and Millipore technologies have a good potential for automation Multiple other methods are available for purifying sequencing reactions and may be used if validated in the laboratory 18 0 Preparing Sequencing Reactions for Electrophoresis 40 The different sequencing instruments may require different preparations of the samples prior to loading them on the gel or the capillary The following chapters describe preparing samples for the capillary sequencers 310 3100 3130 3700 and 3730 and the slab gel sequencer 377 Applied Biosystems For other DNA Sequencers the preparations described may be suitable as well For detailed information refer to the manufacturer s recommendations To prepare purified sequencing reactions for capillary electrophoresis resuspension in formamide or water is possible For ease of use higher reproducibility and higher data quality the water protocol is recommended for the Pr
31. cted and reported Typing results and sequence electropherograms can be printed exported and archived The software is available as a Windows single user version or as a Windows or Linux server client version for use in a network with several workstations The HLA database is continuously updated in line with the latest scientific research of the official IMGT HLA database Instrument Platforms The PCR and sequencing cycle profiles provided in this manual have been used with the Thermal Cyclers from Applied Biosystems GeneAmp PCR Systems 2700 9600 and 9700 They should also work with compatible instruments but may require adjustments of the cycling profile or emulation of the above instruments The Sequencing Kits as well as the SEQUENCE PILOT Allele Identification Software are compatible with all four dye capillary sequencing instruments available Applied Biosystems Capillary 310 3100 3130A 3130 3500 3500 3730 3730xIl GE Healthcare Capillary MegaBACE 500 1000 4000 Beckman Capillary CEQ8000 GeXP Protrans User Manual SBT Version 2010 07 01 7 3 0 Procedure Protrans Sequencing System Separation of Haplotypes DNA Amplification PCR SSP Purification of the separated Haplotypes HLA Class EXON 1 2 3 4 forward and reverse Sequencing the single Haplotypes HLA Class EXON 2 forward and reverse and codon 86TG Purification of the Sequencing Products Running
32. e PCR products to be sequenced An amplification reaction is considered positive if an intensive PCR fragment occurs The Protrans S1 and Protrans Domino Stones PCR product can directly be purified and sequenced without Agarose gel analysis In the Protrans S4 S3 and S2 HLA Sequencing Kits depending on the alleles present in the DNA sample to be investigated one two three or four PCRs can be positive The amplification pattern should be consistent with the specificities listed in the Protrans Attachment Primer Mix Specification Tables Note Itis recommended to select those PCR products for sequencing that have not been generated by overlapping primer mixes If a PCR based separation of alleles is not achieved it is strongly recommended to sequence in both orientations Note The PCR amplification pattern should be in concordance with the specificity table and give a conclusive result If the PCR amplification pattern is inconclusive e g by indicating the presence of more than two alleles then contamination or a variant allele may exist If the negative control excludes contamination sequencing of two PCR products can be performed If the sequencing results indicate identical sequences the other group specific PCR products should be sequenced Note The related positive amplification control should be positive and the related negative amplification control must be negative to exclude contamination Protrans User Manual SBT Version 2010 07 0
33. e aware of this possible limitation and it is strongly recommended to perform low resolution typing by PCR based methods if only a single allele has been identified Mark the positive PCR primer mixes in the Protrans Attachment Select Haplotypes that will be sequenced Type result in Protrans Pipetting Assistant or pipetting scheme form Generate purification plate and sequencing plate with PROTRANS Pipetting Assistant Protrans User Manual SBT Version 2010 07 01 13 0 Protrans Attachment 02 04 05 06 07 08 09 11 12 13 14 15 16 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 01 02 03 04 05 06 07 08 PCR MIX Version PCR MIX ENT Kaes e Le A 23 24 0103 w o A 2433 A 25 26 34 43 66 A 68 69 3401 3405 6602 6603 A 29 31 32 33 74 A 3204 Lex I1 A 29 32 74 w o A 3204 wo A 30 0102 1 eS eT LOT PCR MIX SA HLA C Specificity PCR MIX Cw 02 1511 gt pee aH Cw 06 18 w o Cw 06020102 el 01 03 04 14 18 Cw 02 05 06 08 12 15 16 17 eem LEE puc Lor Liese PCR MIX
34. e supernatant 180x g 20 sec Note Centrifugation is important to ensure complete removal of the degraded products 11 50 80 Ethanol pipet to each well using an 8 channel multi dispensing pipette 12 Spin MicroAmp Optical plate at for 2 000 xg 5 min 13 Immediately remove supernatant by inverting and gently tapping tray on paper towels Place the inverted MicroAmp Optical plate with the paper towels in the centrifuge 14 Spin MicroAmp Optical plate to remove the supernatant at 180x g 20 sec Note Centrifugation is important to ensure complete removal of the degraded products 15 Dry the tray for in dark ambient reactions are light sensitive 15 min 16 10 HPLC water to each well using an 8 channel pipette H Resuspend the sequencing Products by pipetting 3 x up and down 17 90 HPLC water to each well using an 8 channel pipette Close the plate and vortex 18 Spin MicroAmp Optical plate briefly and place on the ABI Support Base Place a Full Plate Septa mat on the tray followed by the 96 Well Plate Retainer and 19 place the complete tray onto the ABI Sequencer Alternative Steps Preparing 125mM Sodium EDTA pH 7 0 1 part 0 5M EDTA and 3 parts HPLC H O 125 mM EDTA pH 7 0 4 H add to the sequencing reactions Single or 8 channel Multistepper Use filter tip 2ul 3 M Sodium Acetate pH 5 2 H add to the sequencing reactions Single or 8 channel Multistepper Use filter tip 16 15 of Hi Di Formamide to each well using an 8
35. f the purfied sequencing reaction must be adapted Denature resuspended samples at 90 for 2 minutes and keep on ice until loading on the gel Protrans User Manual SBT Version 2010 07 01 19 0 Running the Instruments Instrument platforms The Protrans HLA Sequencing Kits are compatible with all four dye sequencing instruments available Follow the manufacturer s instructions for standard runs A read length of 350 bases is sufficient Applied Biosystems Capillary 310 3100 3130 3700 and 3730 Slab Gel 377 GE Healthcare Capillary MegaBACE 500 1000 4000 Beckman Capillary CEQ8 MJ Research Slab Gel BaseStation BaseStation51 For comprehensive information on these instruments and for running other four dye DNA Sequencers refer to the user s manual of the manufacturers Note The Protrans HLA Sequencing Kits have been validated for the Applied Biosystems DNA Sequencers 3100 Genetic Analyzer and 3730 DNA Analyzer Other four dye sequencing instruments should be validated before routine use 20 0 Identifying HLA alleles Protrans Allele Identification Software The final step in sequence analysis consists in the allele assignment using the Protrans Software Sequence Pilot Allele Identification Software JSI medical systems www jsi medisys de This program performs allele identification based on the cDNA sequence database of all HLA class and class exon sequences detects heterozygous positio
36. fere with the sequencing chemistry resulting in lower data quality 14 1 PROTRANS AmpliPur Fast The PROTRANS AmpliPUR Fast PCR purification kit provides a convenient tool for fast and efficient direct purification of PCR products Instruction Binding Buffer vortex and pipet into the PCR products which have to be purified place Protrans Spin filter in the marked Protrans Receiver Tubes 2 0ml PCR product with Binding Buffer pipet in the center of the spin column Protrans Receiver Tubes centrifuge at 10 000 rpm 3 Min place Protrans Spin filter in new marked Protrans Receiver Tubes 1 5ml Elution Buffer EB pipet in the center of the Protrans spin column Incubation at RT 1 Min centrifuge at 6 000 rpm 1 Min 1 150pl Oo Oo Po 14 2 ExoSAP IT Purification Purification of the PCR products with Exonuclease and shrimp alkaline phosphatase enzyme mix EXoSAP IT Degradation of unbound primers and dNTPs Instruction PCR Product Haplotype which has to be purified pipet to defined position on PCR plate After incubation and cooling to room temperatur e the PCR products are ready for sequencing set up 34 Protrans User Manual SBT Version 2010 07 01 14 3 Beads fishing Method An alternative to the ExoSAP IT purification is the use of Agencourt AMPure beads based technology Beckman Coulter 2 3 which fishes the PCR products by attac
37. hing them to the beads and allowing to wash away the residual primers and dNTPs This technique does not need a thermal cycler and can easily be automated The beads purification is compatible with low to high throughput formats using single tubes 96 or 384 well microplates 15 0 Selection of Sequencing Primers The Protrans S4 S3 S2 and Protrans Domino Stones HLA Sequencing Kits are designed to amplify and sequence both alleles separately The Protrans S1 HLA Sequencing Kits are designed to amplify and sequence both alleles simultaneously Note Refer Protrans Attachment Primer Mix Specification Tables for sequencing primer selection In HLA class at least Exons 2 and 3 should be sequenced to get 4 digit HLA typing results In HLA class Il at least Exon 2 should be sequenced to get 4 digit HLA typing results For lower resolution HLA typing results less sequence information may be sufficient If separation of the alleles is achieved 2 Haplotypes it is sufficient to sequence each of the two selected PCR products in a single orientation In HLA class getting complete Exon sequences it is recommended to use the Reverse Sequencing Primers Exon 2 E2R and Exon 3 E3R In HLA class getting complete Exon sequences it is recommended to use the Forward sequencing primers Exon 2 E2F If a PCR based separation of the alleles is not archieved sequencing in both orientations i
38. ifferent Protrans Sequencing Kits in the different Protrans Sequencing Strategies 4 Protrans User Manual SBT Version 2010 07 01 Protrans S3 Sequencing Kits The Protrans S3 sequencing Kits are designed to match the requirements of high sample throughput as well as allele specific sequencing for less ambiguities This is achieved by splitting the Haplotypes before sequencing each Haplotype separately In the Protrans SBT Test kit HLA A B C the DNA will be amplified with 7 Group Specific PCR Amplification Mixes GSA and in addition a Locus specific Amplification Mix LSA In the Protrans SBT Test kit HLA DQB1 the DNA will be amplified with 6 Group Specific PCR Amplifications Mixes GSA and in addition 2 Locus specific Amplification Mixes LSA In the Protrans SBT Test kit HLA DRB1 the DNA will be amplified with 8 Group Specific PCR Amplifications GSA If the GSA reactions do not indicate two separate alleles the LSA reaction must be sequenced This ensures in all cases the recognition of both alleles In the Protrans SBT Test kit HLA class Exons 1 Exon 2 Exon 3 and Exon 4 and in the Protrans SBT Test kit HLA class Exon 2 DRB1 DQB1 and Exon DQB1 are covered The Amplification Primer Mixes are pre pipetted in 8 well PCR Strips and each HLA locus in a different colour In most cases sequence analysis of separately both alleles will be achieved In HLA class special emphasis was put on the complete coverage of Exons 1
39. ipet in a MicroAmp Optical plate always use new filtertips The sequencing products are ready for loading into sequencing instruments Until loading the plates should be stored at 4 8 C protected from light Protrans User Manual SBT Version 2010 07 01 17 2 Ethanol Precipitation 1 80 Ethanol 8 parts Ethanol 10096 and 2 parts HPLC water 2 Spin briefly the 96 well MicroAmp Optical plate with the Sequencing products Take the EDTA NaOAc buffer vortex spin down gently before opening vial and keep on ice Protrans PCR Workstation NaOAC EDTA buffer Teknova cat no S2080 pipet to each sequencing reaction 4 2ul Add the drop to the wall of each well using an 8 channel multi dispensing pipette Note The static can keep the drop at the tip assure dispensing Spin MicroAmp Optical plate briefly 25ul 100 Ethanol pipet to each well using an 8 channel multi dispensing pipette Vortex the plate thoroughly 7 Using a flat support over the tray to prevent any splash e g aluminium plate 30 sec Note Incomplete mixing will result in poor quality data 8 Spin MicroAmp Optical plate at for 2 000x g 30 min 9 Immediately remove supernatant by inverting and gently tapping tray on paper towels Place the inverted tray with the paper towels in the centrifuge 10 Spin MicroAmp Optical plate at to remove th
40. le Allele and Locus specific Sequencing PCR Solution D PSD 2x 1 750 20 Negative control NC ABC 1x 140 20 Protrans Domino Stone HLA C I Haplotype specific Sequencing Allele and Allele Group specific Amplification GSA 1x 1 400 20 PCR Solution L PSL 3x 1 000 20 Protrans S1 HLA C I Locus specific Sequencing EXON 2 4 Locus specific Amplification Primer LSA 1x 360 200 Protrans User Manual SBT Version 2010 07 01 11 Protrans S4 DRB1 Allele and Allele Group specific Sequencing Solution Tubes PCR Solution D PSD 4x 1 750 20 Protrans S3 DRB1 I Allele and Allele Group specific Sequencing PCR Solution D PSD 2x 1 750 20 Negative control NC ABC 1x 140 20 Protrans Domino Stone DRB1 I Haplotype specific Sequencing Allele and Allele Group specific Amplification GSA 1x 1 400 20 PCR Solution L PSL 3x 1 000 20 Protrans S2 DRB1 Allele and Allele Group specific Sequencing PCR Solution D PSD 1x 1 750 206 Protrans S1 HLA DRB E 55 Solution Tubes Volume pl Locus specific Sequencing Locus specific Amplification LSA 1x 360 20 Protrans S1 HLA DRB4 Solution Quantity Volume pl I Locus specific Sequencing Locus specific Amplification LSA 1x 360 20 Protrans S1 HLA DRB5 Solution Quantity Volume pl I Locus specific Sequencing Locus specific Amplification LSA 1x 360 206 P
41. mplified material it is recommended that the work area is strictly partioned as follows Precautionary Pre PCR area measures All work carried out before PCR preparing and storing sample DNA preparing PCR amplification reactions setting up and storing reagents and solutions for DNA isolation and PCR Post PCR area All work carried out after PCR running thermal cyclers and DNA sequencers performing agarose gel electrophoresis preparing and purifying sequencing reactions storing amplified DNA or sequencing reactions Materials and Equipment from the post PCR area must not be taken into the pre PCR area When pipetting in the pre PCR area and for setting up the sequencing reactions tips with aerosol protection filtered tips should be used It is recommended that for each amplified DNA sample a related negative control is performed as an indication of contamination with foreign DNA The following table lists possible causes and solutions for PCR problems Protrans User Manual SBT Version 2010 07 01 43 44 PCR Troubleshooting Table Protrans Problem Possible Cause Solution No PCR No ethidium Secondary staining of the gel in a staining product or bromide in gel bath 1X TBE with 0 5 ug ml ethidium weak PCR bromide remember to add ethidium product bromide prior to pouring the gel Incomplete mixing of Repeat PCR with attention to mixing AmpliTaq Gold and PCR reaction mix Degraded
42. naturation 966 1 Hold Denaturation 96C 10 sec Annealing 506 5 25 Cycles Extension 60 4 min Terminal Cooling 46 Hold Volume 10 pl Step Temperature Time Cycles Denaturation 966 20 Annealing 50T 20 sec 30 Cycles Extension 60 3 min Terminal Cooling 4C Hold Volume 10 pl Note The PCR and sequencing cycle profiles provided in this manual have been used with the Thermal Cyclers from Applied Biosystems GeneAmp PCR Systems 2700 9600 and 9700 They should also work with compatible instruments but may require adjustments of the cycling profile or emulation of the above instruments See Appendix A troubleshooting guide 22 Protrans User Manual SBT Version 2010 07 01 9 0 Specificities of the Amplification Primers 10 0 The method is based on PCR amplifications starting with genomic DNA The PCR amplification reactions are covering at least exons 2 and 3 in HLA class loci and exon 2 in HLA class II loci and have been designed to be specific for a single group of HLA alleles only and a single HLA locus Each of the PCR formulations has been validated against a panel of well characterized cell lines to ensure against non specific amplification and preferential amplification of one allele over another in heterozygote combinations The Protrans SBT Kits are continuously updated The Attachment for each kit is LOT Version and Database specific For interpretation of the amplification results and selection
43. ncing kits are designed to match the requirements of very high sample throughput as well as allele specific sequencing for less ambiguities This is achieved by applying 4 Group Specific PCR Amplifications GSA in parallel covering at least exons 2 and 3 in HLA class loci and Exon 2 in HLA class II loci allowing in many cases sequence analysis of both alleles separately For ease of use e The 4 Group Specific Primer Mixes are pre pipetted twice in a 8 well PCR Strips Test procedure and the Thermocycler program for the Amplification Cycle Sequencing and the Settings of the Sequencer are for all Protrans Sequencing Kits and for all HLA loci identical e Purification of the PCR Products and Sequencing Products are identically for all HLA loci e It is very easy to type different HLA loci of several DNA Samples in parallel e Itis always possible to combine the different Protrans Sequencing Kits in the different Protrans Sequencing Strategies 10 9 Protrans S2 Sequencing Kits pre coated PCR Strips GSA T mixi 10 10 Set up Amplification Protrans S2 Sequencing Kits Documentation of DNA samples with Protrans Pipetting Assistant or pipetting scheme form Place reagents and strips in PROTRANS PCR Workstation 20 C 1 8 well PCR Strip 2 PROTRANS PCR Solution D PSD 3 AmpliTaq Gold DNA Polymerase 4 DNA sample Reaction tube 1 5ml Master Mix for each DNA sample 1 70 pl PCR Solution PS
44. nin E3 20 Electronic Multistepper Rainin EDP3 E3 100 Electronic Multistepper Rainin EDP3 Plus E8 20 Electronic Multistepper 8 channel Multistepper Rainin EDP3 Plus E8 300 Electronic Multistepper 8 channel Multistepper 2 20ul Big Dyes PROTRANS ER amour VE 3 PROTRANS Set up sequencing reaction 10 100ul Sequencing primers PROTRANS 2 20ul PCR products PROTRANS 20 300ul Ethanol precipitation PROTRANS 2 20u1 GP L10F PROTRANS Pipettes filter tips 20 2001 GP L 200F PROTRANS 200 3001 RT L300F PROTRANS Elektrophorese Unit PROTRANS Gel Check or Protrans Quattro Gel Check 4 x 96 Amplificats in 4 Gels in 1 chamber Agarose molecular biology grade 1x TAE TBE Buffer Heating block Microwave oven Ethidium bromide solution 10mg ml WARNING CHEMICAL HAZARD Ethidium bromide is a known mutagen It can change genetic material in a living cell Before using ethidium bromide read the manufacturer s MSDS which gives information on physical characteristics hazards precautions first aid spill clean up and disposal procedures Always wear appropriate protective eyewear clothing and gloves Photograph unit Power supply Transilluminator PCR 8 strips for Loading Buffer PCR reaction plate 96 well and cap strips 8 channel pipette transferring the Loading Buffer into a MicroAmp 96 well reaction plate or MicroAmp strips for mixing the PCR Amplificats with Loading Buffer for loading into the Gel
45. ns as well as mismatches with the sequence database allows manual review or editing of the sequencing data as well as reporting exporting printing and archiving of sequences and results The HLA sequence library is updated with each new Sequence Database release of the HLA informatics group www anthonynolan com HIG and can be downloaded from the www jsi medisys de website The software is availabel as a Windows single user version or as a Windows or Linux server client version This chapter is designed to be a quick reference to help the user through the basic steps involved in performing Sequence Pilot software analysis For more details refer to the Sequence Pilot User s Manual The Sequence Pilot Allele Identification Software is compatible or adaptable to all four dye sequencing instruments available Applied Biosystems Capillary 310 3100 3130 3700 and 3730 Slab Gel 377 GE Healthcare Capillary MegaBACE 500 1000 4000 Beckman Capillary CEQ8 MJ Research Slab Gel BaseStation BaseStation51 Protrans User Manual SBT Version 2010 07 01 41 21 0 Sample Naming Conventions 42 Guideline The Sequence Pilot Software automatically recognizes the locus and exon sequenced as well as the direction of sequencing In order to allow the software for automated joining or pairing of sequencing results that belong together e g forward and reverse sequencing direction of the same PCR product
46. oci The Single Allele or Group Specific Primer Mixes are pre pipetted in 16 well PCR Strips In almost all cases sequence analysis of separately both alleles will be achieved In HLA DRB1 a special emphasis was put on the separation of the DR52 associated HLA DRB1 alleles to ensure unambiguous results in nearly all samples Special emphasis was put on the location of the sequencing primers to ensure complete Exon sequences in both orientations For ease of use The Allele and Allele Group Specific Primer Mixes are pre pipetted in 16 well PCR Strips The colour of the strips are for each HLA locus different The Test procedure and the Thermocycler program for the Amplification for Cycle Sequencing and the Settings of the Sequencer are for all Protrans Sequencing Kits and for all HLA loci and identical The Purification of the PCR Products and Sequencing Products are identically for all HLA loci It is very easy to type different HLA loci of several DNA Samples in parallel It is always possible to combine the different Protrans Sequencing Kits with the different Protrans Sequencing Strategies Protrans User Manual SBT Version 2010 07 01 23 10 1 Protrans S4 Sequencing Kits pre coated PCR Strips HLA A ie 1 at the cut corner csa 1sa HLA C D 15 HLA DRB1 ns 1 at the cut corner 0000 0000 O GSA e controls 24 Protrans User Manual SBT Version 2010 07 01 10 2 Set up
47. of the sequencing primers the Attachment delivered with the kit Version and Database specific must be used exclusively The Protrans HLA Sequencing Kits are continuously updated In order to provide the user with the most recent version of the kit the Primer Mix Specifications are listed as an Attachment in the Primer Mix Specification Tables delivered with each kit Please make sure that the Version of the kit is identical with the Version of the ATTACHMENT the Primer Mix Specification Table in the Testkit PROTRANS S4 Sequencing Kits The Protrans S4 HLA SBT Typing kits are designed to reach a maximal level of allele specific sequencing and in turn the lowest number of ambiguities This is achieved by splitting the Haplotypes before sequencing each Haplotype separately In HLA class I the DNA will be amplified with up to 14 Group Specific PCR Amplifications GSA and in addition a locus specific Amplification LSA in parallel covering Exons 1 Exon 2 Exon 3 and Exon 4 The Single Allele or Group Specific Primer Mixes are pre pipetted in 16 well PCR Strips In almost all cases sequence analysis of separately both alleles will be achieved In HLA class special emphasis was put on the complete coverage of Exons 1 2 3 and 4 to Sort out nearly all ambiguities caused by variations outside Exons 2 and 3 In HLA class II the DNA will be amplified with up to 14 Group Specific PCR Amplifications GSA in parallel covering the complete Exon 2 l
48. onents can be used until the expiry date indicated on the Pre PCR Box and Post PCR Box kit Open packages and Tubes must be closed and stored under the same conditions as closed packages and Tubes and can be used until their expiry date Only components of the same kit Version can be used with each other and only until the marked expiry date 6 0 Precautions and Warning IVD Reagents only for In Vitro Diagnostic use In the US only for research RUO A The PROTRANS Testkits must be performed by well trained and authorised laboratory technicians All reagents should be handled in accordance to good laboratory practice using appropriate precautions In addition handle all patient samples as potentially infectious Do not pipette by mouth All used PCR Cyclerplates should be treated as potentially infectious and should be destroyed according to the valid national guidelines Do not use reagents which are expired See expiration date printed on the label Pre PCR and Post PCR rooms must be strictly separated Use separate pipettes in the Pre PCR area and in the Post PCR area Ethidium bromide used for staining of DNA is a potential carcinogen Always wear protective gloves when handling stained gels Waste management according to national guidelines Wear UV blocking eye protection and avoid direct UV light when viewing or photographing gels See Material Safety Data Sheet MSDS
49. ood should be avoided as heparin has been shown to seriously affect PCR yield The DNA concentration must be 50 100 ng ul diseases and should be handled with precaution Always wear appropriate Working with human blood samples has the potential to transmit infectious protective eyewear clothing and gloves Protrans User Manual SBT Version 2010 07 01 21 8 0 Program Thermocycler Protrans SBT All Protrans HLA Sequencing Kits Protrans S4 S3 S2 S1 and Domino Stones are running with the same Cycling Profiles for PCR Amplification and Cycle Sequencing The program is standardised for HLA Loci Important Note Ramp Rate 1 t s CR Step Temperature Time Cycles Initial Denaturation 956 2 min Hold Denaturation 96C 40 sec Annealing 646 1 min 15 Cycles Extension 726 2 Denaturation 966 20 sec Annealing 60T 1 min 15 Cycles Extension 726 2 Denaturation 966 20 Annealing 56T 1 min 10 Cycles Extension 726 2 Terminal cooling 4C Hold Volume 15 pl 8 2 Purification of PCR Products with ExoSAP IT Step Temperature Time Process 1 Degradation of primers at 2m and dNTPs 2 80T 15 min Degradation of enzymes 3 Ar After incubation and cooling to room temperature the PCR products are ready for sequencing set up Step Temperature Time Cycles Initial De
50. otrans Domino Stone Sequencing Kits Documentation of DNA samples with Protrans Pipetting Assistant or pipetting scheme form Place reagents and PCR strips in PROTRANS PCR Workstation 20 1 Single Tube 8 well PCR Strip or 96 well PCR plate Domino Stones Amplification Primer Mixes for different HLA loci Allele or Allele Group specific PROTRANS PCR Solution L PSL AO m AmpliTaq Gold DNA Polymerase 5 DNA sample Vortex all reagents and spin down with mini table centrifuge Reaction tube 1 5ml Master Mix for 1 DNA sample 1 8 85 pl PCR Solution PSL 2 0 15 pl AmpliTaq Gold DNA polymerase Vortex Master Mix and spin down with mini table centrifuge Master Mix in 1 well of a PCR Strip A 3 ul with electronic Multistepper RAININ 3 5 ul Domino Stone Amplification Primer Mix H in 1 well of a PCR Strip Vortex DNA sample and spin down with mini table centrifuge DNA sample 50 100 in the specific well of S ER the strip to the Domino Stone Close PCR strip Place the strips in the thermocycler and start the PCR amplification program Protrans Domino Stone negative control should be performed for each series of DNAs tested to exclude contamination of the PCR solution and AmpliTag Gold DNA Polymerase 28 Protrans User Manual SBT Version 2010 07 01 10 8 PROTRANS S2 Sequencing Kits The Protrans S2 seque
51. otrans HLA Sequencing Kits Preparing Sequencing Reactions for Capillary Electrophoresis As already desribed in 22 1 and 22 2 dilute the purfied Sequencing Reaction 10 pl Purified Sequencing Reaction with 90 pl HPLC water Seal the plate or tubes appropriately and place them on the autosampler Note Different sequencers may have different fluorescence detection sensitivities and may therefore require different dilutions Note If less or more DyeTerminators are used in the sequencing reaction as specified in 17 the dilution of the purfied Sequencing Reaction must be adapted Note The remaining Sequencing Reaction may be stored as a back up at 4 C for several days in the dark Preparing Sequencing Reactions for Slab Gel Electrophoresis Dry the purified sequencing samples a vacuum centrifuge or in a heating block at 70C 45 min Loading Buffer recrystallized formamide and blue dextran EDTA solution 5 1 prepare 4 yl fresh for each use resuspend the dried Sequencing Sample If spin filtration was used 18 1 skip 5 don t dilute add 4pl Loading Buffer If Etanol precipitation was used 18 2 step 15 don t dilute add 4pl Loading Buffer Note Different sequencers may have different fluorescence detection sensitivities and may therefore require different dilutions Note If less or more DyeTerminators are used in the sequencing reaction as specified in 3 4 1 the dilution o
52. rification of the PCR Products and Sequencing Products are identically for all HLA loci e It is very easy to type different HLA loci of several DNA Samples in parallel e Itis always possible to combine the different Protrans Sequencing Kits in the different Protrans Sequencing Strategies Protrans User Manual SBT Version 2010 07 01 10 4 Protrans S3 Sequencing Kits pre coated PCR Strips HLA A B C GSA 00000000 t mix 1 HLA DRB1 GSA 00000000 T mix 1 HLA DQB1 GSA LSA C amp GO CO OO 00 Ji mix 1 10 5 Set up Amplification Protrans S3 Sequencing Kits Documentation of DNA samples with Protrans Pipetting Assistant or pipetting scheme form Place reagents and strips in PROTRANS PCR Workstation 20 C 1 8 well PCR Strip 2 PROTRANS PCR Solution D PSD 3 AmpliTaq Gold DNA Polymerase 4 DNA sample Vortex all reagents and spin down with mini table centrifuge Reaction tube 1 5ml Master Mix for each DNA sample 1 140 pl PCR Solution PSD 2 1 5 ul AmpliTaq Gold DNA polymerase Vortex master mix and spin down with mini table centrifuge 3 10pl DNA sample 50 100ng ul in the Master Mix Vortex master mix and spin down with mini table centrifuge Master Mix in Protrans 8 well PCR Strip with electronic Multistepper RAININ Close 16 PCR strip Place the strips in the thermocycler and start the PCR amplification program
53. s Contamination of the DNA sample To exclude contamination of the DNA sample re extract DNA from a new source sample Use filter tips User Manual SBT Version 2010 07 01 Sequencing Troubleshooting Sequencing Troubleshooting Table Protrans Sequencing problems may be related to sequences in general or only to heterozygous sequences Problems with heterozygous sequences may be peak shifts or weak heterozygous signals The majority of these anomalies occur in only one sequencing orientation for a certain base position and can be resolved by reviewing data from the other orientation The following table lists possible causes and solutions for general sequencing problems Problem Possible Cause Solution Weak signal Inappropriate Because of variations between instruments strength injection time or adjustments of the injection time and or the injection voltage injection voltage may be needed to get a signal range from 100 2000 relative fluorescent units Too little sequencing Capillary DNA sequencer Increase reaction applied injection time injection voltage or concentration of sequencing reaction Slab gel DNA sequencer Increase loading volume or concentration of sequencing reaction Too strong Inappropriate Because of variations between instruments signal Injection time or adjustments of the injection time and or the strength injection voltage injection voltage may be needed to get
54. s required In order to avoid ambiguities even in heterozygous sequencing due to undefined cis linkages of sequence motifs the use of Sequence Specific Sequencing Primers allows a selective sequencing of only one of the two alleles Together with the result of the heterozygous sequencing an unambiguous cis linkage definition of both alleles can then be obtained Sequence Specific Sequencing Primer HLA DRB1 DR 86TG is presently available for codon 86 selective sequencing Protrans User Manual SBT Version 2010 07 01 35 15 1 Set up Sequencing Reaction The DNA is splitted into 2 Haplotypes HLA class Exon Reverse E2R forward E2F Exon 3 Reverse E3R forward E3F 7 5 Exon 2 Reverse E2R forward E2F aplotype Exon 3 Reverse E3R forward E3F HLA class Il Haplotype 1 Exon 2 Forward E2F reverse E2R Haplotype 1 Exon 2 Forward E2F reverse E2R It is possible to sequence each Haplotype only in one direction 15 2 Set up Sequencing Reaction Only 1 Allele or only the Locus specific Amplification Product LSA HLA class aplotype Boe Exon 3 forward E3F reverse E3R Locus specific Exon 2 forward E2F reverse E2R Amplification LSA Exon 3 forward E3F reverse E3R HLA class Il Haplotype Exon2 forward 2 reverse 2 Codon 86TG Locus specific Amplification LSA Exon 2 forward E2F reverse
55. s specific amplification and sequencing LSA Protrans User Manual SBT Version 2010 07 01 3 2 0 PROTRANS HLA Sequencing System The Protrans Sequencing System is designed to reach a maximum level of allele specific sequencing and in turn the lowest number of ambiguities With the separation of the DNA with specific primer Mixes in two separate Haplotypes ensures the recognition of both alleles in nearly all cases The Protrans Sequencing System allows in HLA class sequencing of Exon 1 to Exon 4 in HLA class sequencing of Exon 2 and codon 86TG Special emphasis was put on the complete coverage of exons 1 2 3 and 4 to sort out nearly all ambiguities caused by variations outside Exons 2 and 3 as well as on the location of the sequencing primers to ensure complete Exon sequences in both orientations Intended use Protrans Sequencing System is easy to use for the application of bone marrow registry typing as well as a diagnostic tool for patient characterization in daily routine Protrans S4 Sequencing Kits The Protrans S4 HLA sBT Typing kits are designed to reach a maximal level of allele specific sequencing and in turn the lowest number of ambiguities This is achieved by splitting the Haplotypes before sequencing each Haplotype separately In HLA class I the DNA will be amplified with up to 14 Group Specific PCR Amplifications GSA and in addition a locus specific Amplification LSA in parallel covering Exons 1
56. sult files table Selects pairs forward and reverse or joins different exons the result files that belong together and have to be analyzed together Process Note This is done automatically if the Sample Naming Conventions have been followed Generates a combined sequence for paired forward and reverse sequences Starts allele identification for paired or joined result files by comparing the result sequences with the HLA sequence library Analysis Menu 2 Mark the joined result files in the Worklist and select Sequence in the analysis window Process Displays the electropherograms and result of allele assignment in the Data Analyzer window 3 Check the results using the electro pherogram options Process Edits and re analyzes the result sequences if required 4 Approve final analysis Process Validates allele identification saves data in the result database transfers the result into the local LIS if desired and prints the final report for documentation 23 0 Trouble shooting Guide PCR PCR is an extremely sensitive method which can efficiently amplify even the Troubleshooting smallest amount of DNA It follows from this that even traces of contaminating DNA in a sample can be amplified in the PCR reaction and falsify the test result One particular source of contamination is amplified DNA coming into contact with samples which are still to be amplified To avoid contamination with a
57. tch the requirements of very high sample throughput This is achieved by Locus Specific PCR Amplification LSA covering exons 2 and 3 in HLA class loci leaving ambiguities due to variations outside Exon 2 and 3 unsolved A special emphasis was put on the generation of short PCR products to increase robustness in case of DNA of lower quality For ease of use the Locus Specific Amplification Mix is ready for use The PCR Amplification set up for Protrans S1 and Protrans S1 swift Sequencing Kits is identical 10 12 Set up Amplification Protrans S1 Sequencing Kits Documentation of DNA samples with Protrans Pipetting Assistant or pipetting scheme form Place reagents and PCR strips in PROTRANS PCR Workstation 20 1 Single Tube 8 well PCR Strip or 96 well PCR plate Domino Stones Amplification Primer Mixes for different HLA loci Allele or Allele Group specific PROTRANS PCR Solution D PSL AO m AmpliTaq Gold DNA Polymerase 5 DNA sample Vortex all reagents and spin down with mini table centrifuge Reaction tube 1 5ml Master Mix for 1 DNA sample 1 12 ul S1 LSA Mix 2 0 15 pl AmpliTaq Gold DNA polymerase Vortex Master Mix and spin down with mini table centrifuge Master Mix in 1 well of a PCR Strip with electronic Multistepper RAININ Vortex DNA sample and spin down with mini table centrifuge DNA sample 50 100ng ul in the specific
58. te Exon sequences in both orientations Protrans Sequencing Kits Protrans S1 swift REF 2101 and 2102 are a variant of Protrans S1 Kits These Kits are designed to match the requirements of very high sample throughput This is achieved by Locus Specific PCR Amplification LSA covering Exons 2 and 3 in HLA class loci leaving ambiguities due to variations outside Exon 2 and 3 unsolved A special emphasis was put on the generation of short PCR Products to increase robustness in case of DNA of lower quality For ease of use The Group Specific Primer Mix is in a single Tube e Test procedure and the Thermocycler program for the Amplification Cycle Sequencing and the Settings of the Sequencer are for all Protrans Sequencing Kits and for all HLA loci identical e The Purification of the PCR Products and Sequencing Products are identically for all HLA loci e It is very easy to type different HLA loci of several DNA Samples in parallel e Itis always possible to combine the different Protrans Sequencing Kits in the different Protrans Sequencing Strategies 6 Protrans User Manual SBT Version 2010 07 01 Primer Mix Specificities and Specification Tables The Protrans HLA Sequencing Kits are continuously updated In order to provide the user with the most recent version of the kit the Primer Mix Specifications are listed as an Attachment in the Primer Mix Specification Tables Please make sure that the version of the kit is identical wi
59. th the version of the Primer Mix Specification Table PCR Amplification The method is based on PCR amplifications starting with genomic DNA The PCR amplification reactions are covering at least Exons 2 and 3 in HLA class loci and Exon 2 in HLA class II loci and have been designed to be specific for a single group of alleles only Each of the PCR formulations has been validated against a panel of well characterized cell lines to ensure against non specific amplification and preferential amplification of one allele over another in heterozygous combinations Sequencing After PCR clean up the positive Group Specific Amplification reactions are used as templates for direct sequencing at least Exons 2 and 3 in HLA class loci and Exon 2 in HLA class II loci The PCR templates have been optimized for the use of dye terminator cycle sequencing chemistries to generate the allele typing information Allele Assignment The final step in sequence analysis consists out of the allele assignment using the SEQUENCE PILOT allele identification Software This program performs allele identification allows manual reviewing or editing of the sequencing data as well as reporting exporting and archiving of sequences or results SEQUENCE PILOT E allele identification software enables quick and easy allele assignment The software allows manual reviewing or editing of the sequencing data of Exon 1 to Exon 8 Heterozygous positions as well as mismatches are dete
60. tion PROTRANS Domino Stones Sequencing Kits 27 10 8 PROTRANS S2 Sequencing Kits 28 10 9 PROTRANS S2 Sequencing Kits pre coated PCR Strips 28 10 10 Set up Amplification PROTRANS S2 Sequencing Kits 28 10 11 PROTRANS S1 Sequencing Kits 29 10 12 Set up Amplification PROTRANS S1 Sequencing Kits 29 11 0 Analysis of PCR Amplification results Determing the Haplotypes 30 12 0 Documentation of positive Reactions Assignment of Haplotypes 30 13 0 PROTRANS Attachment 32 14 0 Purification of the Haplotypes positive PCR Products 33 14 1 PROTRANS AmpliPur Fast 33 14 2 ExoSAP IT Purification 33 14 3 Beads fishing method 34 15 0 Selection of Sequencing Primers 34 15 1 Set up Sequencing Reaction 2 Haplotypes 35 15 2 Set up Sequencing Reaction only 1 Haplotype or a locus specific amplification LSA 35 16 0 Set up Sequencing Reaction 36 17 0 Purifying sequencing Reactions 37 17 1 PROTRANS DyePUR 37 17 2 Ethanol Precipitation 38 17 3 Alternative purification methods 39 18 0 Preparing Sequencing Reactions for capillary electrophoresis 39 19 0 Running the Instruments 40 20 0 Identifying HLA alleles PROTRANS Allele Identification Software 40 21 0 Sample naming conventions 41 22 0 Performing Allele Identification 42 23 0 Trouble shooting Guide 42 Protrans User Manual SBT Version 2010 07 01 Protrans Sequencing Testkits SBT Protrans S4 Single Allele and Locus Specific Sequencing System
61. well of 4 3 pl the strip Close PCR strip Place the strips in the thermocycler and start the PCR amplification program Protrans S1 HLA Sequencing Kit negative control should be performed for each series of DNAs tested to exclude contamination of the PCR solution and AmpliTaq Gold DNA Polymerase 30 Protrans User Manual SBT Version 2010 07 01 11 0 Analysis of PCR Amplification results Determing the Haplotypes The PCR products are identified by fluorescent agarose gel electrophoresis followed by UV detection of the PCR bands 1 Preparation of 296 Agarose gel 2 1x TBE oder 1x TAE buffer 0 2 ug ml ethidium bromide 3 2 ul Loading Buffer dispense with Multi Stepper in a PCR strip or 96 well PCR plate 4 5 ul PCR amplification reaction mix each PCR amplification reaction with 2ul loading buffer Use a 8 channel pipette compatible with microplate formats 5 5 ul of this Mixture load directly on the gel with the same tips e g use PROTRANS Quattro Gel Check system 1 chamber 4 Gels 96 slots 6 Run the gel in 1X TBE at 20 V cm for 5 20 min For example with the PROTRANS Gel Check electrophoresis chamber a run at 200 V for 5 min 12 0 Documentation of positive Reactions Assignment of Haplotypes Photograph the PCR amplification pattern and record the results on a copy of the Protrans Attachment the Primer Mix Specification Tables provided with each kit and determine th

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