HLA-DRB BigDye™ Terminator Sequencing
Contents
1. 100 uL each tube Five tubes of recrystallized formamide 0 5 mL each One tube of blue dextran EDTA solution 1 0 mL Table 1 Reagents Supplied by the User Reagent Source ABI PRISM dRhodamine Matrix Standards Kit Applied Biosystems P N 403047 Agarose gel loading buffer MLS Table 1 Reagents Supplied by the User continued Reagent Source Agarose molecular biology grade MLS Boric acid MLS Deionized water MLS Disodium ethylenediaminetetraacetic acid dihydrate NagEDTA 2H5O Sigma P N E4884 or Gibco BRL P N SS7SUA DNA size ladders 1 ug L 100 bp or 250 bp Gibco BRL P N 15628 050 10596 013 Ethanol absolute MLS Ethanol anhydrous Kodak IBI P N IB15720 Ethidium bromide Sigma P N E1510 or MLS Sodium acetate 3 M pH 4 6 Applied Biosystems P N 400320 Tris base molecular biology grade MLS Tris HCl molecular biology grade MLS For the ABI PRISM 310 Genetic Analyzer Only 10X Genetic Analyzer Buffer with EDTA Applied Biosystems P N 402824 Performance Optimized Polymer 6 POP 6 Applied Biosystems P N 402837 For the ABI PRISM 377 DNA Sequencer Only Ammonium persulfate Kodak IBI P N 1B70080 Long Ranger Gel Solution 50 FMC P N 50611 Long Ranger Singel pack Type 377 36 cm WTR FMC P N 40691 N N N N Tetramethylethylenediamine TEMED Kodak IBI2. P N IB70120 Urea Kodak IBI P N IB72064 Table 2 Equipment Supplied by the User Item Source ABI PRISM 377 DNA Sequencer or ABI PRISM 310 Genetic Analyzer Applied Biosystems ABI PRISM DNA Sequencing Analysis Software v 2 1 1 v 2 1 2 or v 3 0 Applied Biosystems GeneAmp PCR System 2400 or 9600 Applied Biosystems Heating block MLS MatchMaker Allele Identification Software v 1 0 includes MatchMaker MatchTools and MTNavigator software Applied Biosystems P N M0024 MicroAmp 2400 or 9600 Base Applied Biosystems P N N801 0531 N801 5531 MicroAmp 2400 or 9600 Tray Retainer Set Applied Biosystems P N N801 0530 N801 5530 MicroAmp 2400 or 9600 Full Plate Cover Applied Biosystems P N N801 0550 N801 5550 MicroAmp caps 8 or 12 strip Applied Biosystems P N N801 0535 N801 0534 MicroAmp Optical 96 Well Reaction Plate Applied Biosystems P N N801 0560 MicroAmp Reaction Tubes 0 2 mL or strip tubes Applied Biosystems P N N801 0533 N801 0580 Multichannel pipettor MLS Pipettes and tips MLS Spin Column Centri Sep 1 mL Princeton Separations P N CS 901 Outside North America Applied Biosystems P N 401763 P N 401762 Tabletop centrifuge with 96 well microtiter tray adaptor MLS Vortexer MLS Table 2 Equipment Supplied by the Use
3. c Add 2 5 uL of genomic template AmpliTaq Gold DNA Polymerase mixture to the PCR mixes Positive Control Reactions DRB ALL Add 2 5 uL of the genomic template AmpliTaq Gold DNA Polymerase mixture to a 0 2 mL MicroAmp PCR tube containing 10 uL of the DRB ALL PCR mix To perform low resolution SSP PCR continued Step Action Negative Control Combine 1 0 uL of AmpliTaq Gold Reactions DNA Polymerase with 40 uL DNA DRB ALL Dilution Buffer Add 2 5 uL of DNA Dilution Buffer AmpliTaq Gold DNA Polymerase mixture to a 0 2 mL MicroAmp PCR tube containing 10 uL of DRB ALL PCR Mix Note Using the DRB ALL PCR as a positive or negative control is up to the individual user If detecting contamination is critical then using the DRB ALL as a negative control is appropriate If it is important to ensure that the DNA sample will in fact work in the amplification reactions then it may be more appropriate to use the DRB ALL as a positive control Note An indicator dye may be used by adding 1 2 uL of a 25 mM solution of cresol red dye to the genomic template AmpliTaq Gold mixture The genomic template AmpliTaq mixture has been added to the PCR reactions if the final solutions have slight red tints Place the tubes in a 96 well or 24 well tube tray retainer depending on the thermal cycler to be used A suggested tube arrangement for four DNA specimens in a 96 well tray is shown below Each ro
4. 3 buttons C Use the default settings of 2000 for Start at and 1500 for Points 3 Click New File name the matrix appropriately e g HLA BDT and save it to the ABI Folder in the System folder Click T7 Terminator Matrix 5 Click OK The message Make matrix successfully completed appears 62 Complete the Other Matrices To generate a matrix continued Step Action 6 If an error message occurs modify the default values in the Make Matrix dialog box Make a new matrix Note If you get an error message and the software will not make a matrix you may have designated the wrong files It is possible though rare that the signal is too weak to make a matrix If the signal is inadequate to make a matrix rerun the standard or use another dRhodamine BigDye chemistry sequencing run Repeat steps 1 5 on page 62 for making the Dye Primer Matrix and the Taq Terminator Matrix of the instrument file These two other matrices are needed by the sequencing analysis software to analyze a BigDye Terminator sequencing sample Be sure to select Update File instead of New File when repeating step 3 The end result is a complete instrument file that can be used for analyzing Big Dye and dRhodamine chemistries 63 Evaluate Matrix Quality To evaluate the quality of the matrix Step Action 1 Launch the Sequencing Analysis software 2 In the Sample Manager add the
5. 7 9 1 8 12 12 a za 7 10 1 9 8 12 8 8 12 12 x 1 10 8 12 8 9 B 2 31 6 8 12 8 10 2 4 x 8 12 12 9 B 2 7 8 12 12 10 x 2 8 12 8 z 9 10 2 8 12 12 2 9 2 10 45 Appendix C Troubleshooting Guide Overview The protocol and the HLA DRB BDT Sequencing Based Typing Kit will provide data that allow high resolution allele assignments However certain problematic situations may be encountered as detailed in the tables below Included in each table are descriptions of the problem possible causes and suggestions for resolving the problem PCR Sequencing Symptom Possible Cause Action No PCR Inappropriate cycling Check the protocol and confirm product or parameters the correct method was used weak product DNA not quantitated correctly Re quantitate DNA and adjust to 10 ng uL Degraded DNA Evaluate on agarose gel and re extract the DNA if necessary PCR inhibitors in the genomic DNA Re extract genomic DNA using one of the recommended methods see Blood Collection and DNA Sample Preparation on page 14 Symptom Possible Cause Action Weak signal Inefficient Re sequence and precipitate strengths precipitation of referring to details in Purifying sequence ladder Sequencing Reactions on page 24 Sequence ladder not denatured properly Re sequence
6. PCR Amplifications IMPORTANT This kit should be used in accordance with guidelines established by the Committee for Quality Assurance and Standards of the American Society for Histocompatibility and Immunogenetics 1 The genetic complexity of the DRB region of the human major histocompatibility complex MHC has required development of molecular typing techniques with increasing levels of resolution Methods such as Polymerase Chain Reaction Sequence Specific Oligonucleotide Probe PCR SSOP or Sequence Specific Primer PCR SSP PCR are not always able to discriminate among the approximately 200 recognized alleles at DRB1 To address this need Applied Biosystems has developed a system that combines a low resolution SSP PCR followed by high resolution allele typing using automated DNA sequencing The low resolution SSP PCRs are based on allele group specific motifs in the first hypervariable region of exon 2 The following allele groups and genes are amplified with the eleven specific PCR mixes provided DR1 DR7 DRB3 DR2 DR8 12 DRB4 DR3 11 6 DR9 DRB5 DR4 DR10 In addition to the group specific amplification mixes a control mix is also provided that amplifies all DRB alleles and genes DRB ALL This mix can be used as either a positive or negative amplification control These PCRs have been optimized for use with AmpliTaq Gold DNA Polymerase to ensure specific and efficient reactions 1 1996 ASHI Standa
7. PRISM 310 Genetic Analyzer User s Manual or the ABI PRISM 377 DNA Sequencer User s Manual 47 MatchTools Software Symptom Possible Cause Action Samples not Sample names do not Use the Group these files grouped match command in the MatchTools Worksheet menu Reverse Too much sequence Re analyze with Sequence sequence not automatically reverse complemented by MatchTools software beyond end of PCR product Analysis software process only to the end of the sequence Heterozygotes not identified by MatchTools software Base incorporation anomaly See known anomalies in Table 3 on page 49 Manually edit in MTNavigator software and re submit Allele type not assigned to good quality data Incorrect allele library selected Refer to HLA DRB Allele Libraries on page 42 Anomalies Table Based upon sequences generated with this kit reproducible anomalies have been observed at some base positions The table below summarizes and gives examples of those anomalies that have been encountered and may be useful for interpretation editing of the sequencing data The majority of anomalies occur in only one sequencing orientation for each base position and can be resolved by reviewing data from the other orientation Table 3 Anomalies Heterozygote Sequence Combination Correct InitialBase Nature of Position Codon Orientation2 1s
8. categories to be customized such as Basecaller ABI CE1 Peak 1 Location Same as Start Point Dye Set Primer file 310 HLA DRB BDT and Instrument file Repeat steps 1 3 for each sequence file Start analysis Review the newly analyzed sequences to ensure that the correct analysis parameters were specified Note If you encounter problems with the sequence data generated refer to the AB PRISM DNA Sequencing Analysis Software User s Manual or the Troubleshooting Guide in Appendix C on page 46 35 Recommendations for ABI PRISM 377 DNA Sequencer Users Overview Gel Formulation Preparing and Loading Samples If you have not previously used the dRhodamine based sequencing chemistries the run module and dye set primer mobility file will need to be installed and the instrument matrix files made The run module and dye set primer mobility file are provided on the HLA DRB BDT Supplemental Diskette For more information on making the instrument matrix files see Appendix D on page 60 Note For more information on basic operation and safety see the ABI PRISM 377 DNA Sequencer User s Manual and the ABI PRISM 377 DNA Sequencer Safety Summary Prepare a 36 cm well to read 0 2 mm thick 5 Long Ranger Gel For details see the User Bulletin Improved Gel Formulations for Extended Sequencing Read Lengths P N 4303614 WARNING CHEMICAL HAZARD Long Ranger Gel Solution contains Acrylamide and
9. considered an endpoint Return to the Sample Manager Sample File Queue window Highlight the name of the file just inspected and enter the information for the Start Point and Stop Point Enter information for other categories to be customized such as Peak 1 Location Same as Start Point Dye Set Primer file 377 HLA DRB BDT and Instrument file Repeat steps 1 3 for each sequence file Start analysis Review the newly analyzed sequences to ensure that the correct analysis parameters were specified Note If you encounter problems with the sequence data generated refer to the AB PRISM DNA Sequencing Analysis Software User s Manual orthe Troubleshooting Guide in Appendix C on page 46 Assignment of HLA DRB Allele Types to Sequence Files Overview Using MatchTools Software for Preliminary Analysis Each sequence data file is submitted to MatchTools Software for DRB1 DRB3 DRB4 or DRB5 allele determinations Refer to the MatchMaker Allele Identification Software User s Manual for more information During a preliminary analysis the MatchTools software performs the following functions Pairs sequences having the same sample name These are typically a forward and a reverse sequence from a single PCR product Determines the orientation of each sequence within the pair Compares paired sequences with each other and generates a consensus sequence Compares the consensus sequence a
10. displayed in the injection list to the right of each sample Inj Secs Inj kV Run kV Run C Run Time 30 1 0 15 0 50 28 For best results the signal strengths of the analyzed data should range from 100 1000 relative fluorescence units RFU Because of normal variations among instruments and other factors some adjustments may be needed to modify the signal strength Increase or decrease the injection time and or injection voltage appropriately Injection times greater than 60 seconds are not recommended Performing Choose which data files to analyze then analyze and print them using Fluorescent Signal the Sequencing Analysis Software Analysis Setting the Stop Point in Analysis Parameters Step Action 1 Launch the Sequencing Analysis software and add data files to be processed Open the data file and view the Raw Data Set the stop point to be at the end of the sequence run If too many bases are processed the excess sequence will make it difficult for the MatchTools software to properly define the sequence orientation Sequence Analysis version 3 0 users may want to set the endpoint in the Basecaller settings to 8 Ns in 10 Be aware that any sequence region containing 8 Ns in 10 bases will be considered an endpoint Return to the Sample Manager Sample File Queue window Highlight the name of the file just inspected and enter the information for the Stop Point Enter information for other
11. solely for research and other uses that are not unlawful No other license is granted expressly impliedly or by estoppel For information concerning the availability of additional license to practice the patented methodologies contact Amersham Life Science Inc Vice President Regulatory Affairs P O Box 22400 Cleveland Ohio 44122 Patents are pending in countries outside the United States ABL Applied Biosystems BigDye MatchMaker MatchTools MTNavigator and POP 6 are trademarks and ABI PRISM and its design MicroAmp and Applied Biosystems are registered trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries AmpliTaq AmpliTaq Gold and GeneAmp are registered trademarks of Roche Molecular Systems Inc All other trademarks are the sole property of their respective owners Contents Introductions ecce ESE EPOD EINE DE De BARE S BRD 1 OVGIVICW 3 ic eens hs ot ERR e eg RO RC gea ees eph cu ae 1 SSP PCR Amplifications 2 0 0 0 0 0 eee eee eee 1 Sequencing SSP PCR Fragments 0 00 00 erneer 2 Allele Assignment 0 0 0 0 cece eee RII 2 Instrument Platforms e a c eee eee A 2 Materials and Equipment 00 0 0 eee eee eee 3 Reagents Included 1 0 0 cee eee eee een eens 3 Reagents and Equipment Not Included 0045 3 Safety Information 0 0 cece cee eee eee 6 Technical Support sepe doa ete ahh ole b EPA Oh eae EE 7 To Rea
12. 10 Genetic Analyzer Users Overview Capillary Preparation Preparing and Loading Samples If you have not previously used the dRhodamine based sequencing chemistries the run module and dye set primer mobility file will need to be installed and the instrument matrix files made The run module and dye set primer mobility file are provided on the HLA DRB BDT Supplemental Diskette For more information on making the instrument matrix files see Appendix D on page 60 Note For more information on basic operation and safety see the ABI PRISM 310 Genetic Analyzer User s Manual and the ABI PRISM 310 Genetic Analyzer Site Preparation and Safety Guide To sequence on the ABI PRISM 310 instrument use a 47 cm green dot capillary and Performance Optimized Polymer 6 POP 6 IMPORTANT To ensure adequate signal strength perform the steps below in the exact order they are given To prepare and load the samples from a MicroAmp 9600 tray Step Action 1 Resuspend the DNA pellets in 15 uL of the recrystallized formamide provided in the HLA Control and Loading Buffer Module WARNING CHEMICAL HAZARD Formamide is a known teratogen It can cause birth defects Obtain a copy of the MSDS from Applied Biosystems Web site or Fax on Demand see Technical Support on page 7 Wear appropriate protective eyewear clothing and gloves Wash thoroughly after handling formamide IMPORTANT Use only the recrystallized formamide sup
13. HLA DRB BigDye Terminator Sequencing Based Typing Kit Protocol For Research Use Only Not for use in diagnostic procedures Applied KS Biosystems Copyright 2001 Applied Biosystems For Research Use Only Not for use in diagnostic procedures Printed in the U S A Notice to Purchaser Limited License The purchase of this product allows the purchaser to use it for amplification and detection of nucleic acid sequences for the HLA DRB gene except as stated herein For research and development a license under U S Patents 4 683 202 4 683 195 4 965 188 and 5 075 216 or their foreign counterparts owned by Hoffmann La Roche Inc and F Hoffmann La Roche Ltd Roche has an up front fee component and a running royalty component The purchase price of the HLA DRB BigDye Terminator Sequencing Based Typing Kit P N 4305213 4305026 or 4305027 includes limited nontransferable rights under the running royalty component to use only this amount of the product to practice the Polymerase Chain Reaction PCR and related processes described in said patents for the research and development activities only of the purchaser and only when this product is used in conjunction with a thermal cycler whose use is covered by the up front fee component Rights to the up front fee component must be obtained by the end user in order to have a complete license to use this product in the PCR process for research and development activities T
14. I 0 1 mM of EDTA pH 8 0 4 Quantitate and resuspend purified DNA in DNA dilution buffer to a concentration of 10 ng L 5 Store at 2 6 C a Miller S A Dykes D D and Polesky H F 1988 A Simple Salting Out Procedure for Extracting DNA from Human Nucleated Cells Nucleic Acids Research 16 1215 b Proceedings of the Eleventh International Histocompatibility Workshop and Conference Yokohama Japan November 6 13 1991 15 Low resolution SSP PCR DRB Typing 16 Overview The low resolution DRB typing is based on the SSP PCR results generated with the reagents in the HLA DRB Amplification Module For each DNA sample to be tested 12 separate PCR amplifiications are performed IMPORTANT These manipulations must be performed in an environment free of contamination by human DNA or PCR amplicons such as a separate clean room or biological containment hood Vortex all reagents briefly before using Low resolution SSP PCR To perform low resolution SSP PCR Step Action 1 Prepare samples at room temperature as follows IF preparing THEN Allele Group specific Reactions DRB1G1 DRB1G2 DRB1G3 11 6 DRB1G4 DRB1G7 DRB1G8 12 DRB1G9 DRB1G10 DRB3 DRB4 and DRB5 a Add 1 0 uL AmpliTaq Gold DNA Polymerase stock solution at 5 U uL to 40 uL genomic template 10 ng uL b Add 10 uL of each Allele Group specific PCR Mix to 0 2 mL MicroAmp PCR tubes
15. Keep the Sample Name to 26 characters or less IMPORTANT If you do not separate the sequence direction from the Sample Name with the left hand square bracket the MatchTools software will not correctly group the forward reverse sequences of each SSP PCR The ABI PRISM 310 Collection Program allows the user to set up default file names of each injection as preferences Standardized names attached to the sample name can be created and saved Step Action 1 Open the ABI PRISM 310 Collection Software Under the Window menu choose Preferences 2 3 From Preferences choose Default File Names 4 For the Sample File fill in the three fields as follows a Choose tube numbers in the left pull down window b Click in the middle field and type in lt gt option 8 key stroke c Choose sample name in the right pull down window 5 Click OK The appropriate dye set primer mobility file is 810 HLA DRB BDT supplied on the HLA DRB BDT Supplemental Diskette Choose the correct instrument matrix file 33 Filling in the Injection List Signal Strength Once the sample sheet is brought in to the Injection List choose the run module 310 Run Module HLA DRB BDT for every injection This module file is supplied on the HLA DRB BDT Supplemental Diskette Place it in the Modules folder inside the 310 Collection Software folder When this run module is chosen the following information is
16. PCR product to 6 uL of DNA Dilution Buffer 2 To separate tubes plate wells add 8 uL of each of the Ready Reaction Mixes 3 To each of the Ready Reaction Mixes in the tubes plate wells add 2 uL of diluted PCR product 4 Seal the tubes with MicroAmp caps or cover the plate with a MicroAmp 2400 or 9600 Full Plate Cover 5 Centrifuge briefly in a benchtop centrifuge to combine the PCR product and Ready Reaction Mix at the bottom of the tubes plate wells 6 Place the tubes plate in a thermal cycler 23 Purifying Sequencing Reactions To perform the sequencing reactions continued Step Action 7 Do 1 cycle of the following Rapid thermal ramp to 96 C 96 C for 10 seconds 8 Repeat the following for 20 cycles 96 C for 10 seconds Rapid thermal ramp to 50 C 50 C for 10 seconds Rapid thermal ramp to 60 C 60 C for 2 minutes 9 9 9 9 9 Rapid thermal ramp to 4 C and hold until ready to purify the sequencing reactions Following cycle sequencing the reactions are purified and concentrated to remove unincorporated dye terminators that can appear as artifacts in the sequencing data Three methods have been validated for recovering the sequencing ladders following the cycle sequencing reactions For ease of use as well as increased throughput we recommend the ethanol precipitation procedure using the 96 well format For those laboratories that do
17. R extra A shoulder Complement from following A 2 Reverse E D g n G base 220 codon 74 anomalous A mobility 221 74 Forward 377 0801 1201 Y T low C Reverse incorporation Complement 310 1 Forward t ix E y 6 6 base 221 codon 74 low C x 56 Table 3 Anomalies continued base 255 codon 85 anomalous T mobility bases 257 258 codon 86 incomplete G resolution Heterozygote Sequence Combination Correct InitialBase Nature of Position Codon Orientation 1st Allele 2nd Allele Base Call Call Anomaly 256 86 Reverse codon 86 T K anomalous T Complement hetero mobility see zygotes figure for codon 86 below 257 258 86 Reverse codon 86 KK KT incomplete Complement hetero resolution of G zygotes peaks Forward T t B K K B n G 57 Table 3 Anomalies continued Heterozygote inationb n Sequence Combination Correct InitialBase Nature of Position Codon Orientation 1st Allele 2nd Allele Base Call Call Anomaly 261 87 Reverse All constant G R excess A signal Complement position 2 Reverse G A g B d T base 261 codon 87 excess A signal 267 89 Forward All constant C Y extra T shoulder position from previous T l Reverse T T D A C base 267 codon 89 anomalous T mobility a In this table Reverse Co
18. RB1G7 DRB1G8 12 DRB1G9 DRB1G10 DRB3 DRB4 DRB5 DRB ALL o ea So a Record the electrophoresis results on a form like the one in Appendix A on page 44 Compare the results with the table in Appendix B on page 45 Note An amplification reaction is considered positive if an SSP PCR fragment has an intensity comparable to either the 600 bp fragment of the 100 bp ladder or the 1000 bp fragment of the 250 bp ladder 21 Results as Control Reactions 4 Only one or two of the DRB1 PCRs should be positive If three or 22 Low resolution Considerations to keep in mind while reviewing the low resolution results include the following more DRB1 amplifications are positive then contamination or a PCR problem may exist If none is positive then there may be a problem with the sample the reagents and or the thermal cycler The positive DRB1 PCRs should be consistent with the DRB3 DRBA and DRB5 results based on known linkage patterns These patterns are detailed in the table in Appendix B on page 45 For example refer to the figure in step 6 on page 21 Note that DRB1G4 and DRB1G7 are positive and that DRB4 is also positive Known exceptions to these rules include the few alleles that do not amplify in the expected allele groups as listed below DRBiG8 2 DRB1G4 1105 1122 1317 1410 1404 1415 1428 1431 Other DRB haplotypes may have an unusual amplification pattern If a combination is observed th
19. a matrix Step Action 1 Open the Run folder and highlight the sample file by clicking on the name 2 Open the File menu and choose Duplicate A duplicate is created called Sample file copy 3 Open the File menu and choose Duplicate two more times to create Sample file copy 2 and Sample file copy 3 61 View Matrix Run Use the Data Utility software to view raw data of the matrix runs Raw Data Step Action 1 Launch the Data Utility software 2 Under File choose Open 3 Search for the Run Folder containing the four sample files one original and 3 duplicates 4 Click on the first sample The corresponding open windows are Raw data Analyzed EPT Information 5 Verify that the baseline is stable and peak heights are above 100 fluorescent units in the raw data 6 Choose a scan point e g 2000 that occurs after the first peaks though still in the beginning of the run Generate the The matrix standards are analyzed mathematically to create matrices Terminator Matrix To generate a matrix Step Action 1 From the Utilities menu select Make Matrix The Make Matrix dialog box appears 2 Designate the files for C A G and T data Example a Click the C button then choose Sample file in the Run Folder b Dothe same for the A Sample file copy G Sample file copy 2 and T Sample file copy
20. ample File Step Action 4 For the Sample File fill in the three fields as follows a Choose lane numbers in the left pull down window b Click in the middle field and type in lt gt option 8 key stroke c Choose sample name in the right pull down window 5 Click OK The appropriate dye set primer mobility file is 877 HLA DRB BDT supplied on the HLA DRB BDT Supplemental Diskette Choose the correct instrument matrix file Loading the samples Step Action 1 Load the appropriate volume of each sample into a separate lane as previously designated in the sample sheet 2uL for a 24 lane gel 1 5 uL for a 36 lane gel IMPORTANT Load samples into alternate lanes for example lanes 1 3 5 Prerun samples into the gel for 2 minutes Pause the electrophoresis prerun Load the remaining samples into alternate lanes for example lanes 2 4 6 ABI PRISM 377 Run Parameters Performing Fluorescent Signal Analysis Prior to collecting data enter the settings for each of the electrophoresis parameters Comb Matrix Run Module 377 Run Module HLA DRB BDT Well to Read Distance 36 cm and Sample Sheet The run parameters in the 377 HLA DRB BDT run module are Voltage 1 5 kV Temperature 51 C Time 4 hours Filter set E Note For information about general data collection see the AB PRISM 377 DNA Sequencer User s Manual Foll
21. at is not consistent with the table on page 45 then Record your observations Check your results carefully after sequencing and allele assignment Consult the latest allele sequence layouts and scientific literature For example some DR alleles are on haplotypes that lack the DRB5 gene Forward and Reverse Sequencing Overview Performing Sequencing Reactions High resolution allele typing is based on sequencing the positive SSP PCR reactions This provides sequence for exon 2 in both the forward and reverse orientations The following section describes the steps involved in preparing forward and reverse sequencing reactions from a single SSP PCR amplicon There is no requirement for PCR purification in this procedure the amplicon is simply diluted 1 3 in DNA Diluent buffer or distilled water Depending on the number of positive reactions in the low resolution step the total number of Forward and Reverse sequencing reactions will be between two and eight The sequencing is performed using Ready Reaction mixes that include the sequencing primer and BigDye Terminator Mix Note Use MicroAmp Reaction Tubes or a MicroAmp Optical 96 Well Reaction Plate Note Perform thermal cycling using the correct method Set the sample volume to 10 pL To perform the sequencing reactions Step Action 1 Dilute PCR product 1 3 in DNA Diluent For sufficient sample to sequence in both directions add 2 uL of
22. ation consists of the Applied Biosystems and Celera Genomics businesses Printed in the USA 7 2001 Part Number 4305024 Rev 2
23. ch Us On the World Wide Web 0 eee eens 7 To Reach Us by Telephone or Fax 00 00 0000 7 Hours for Telephone Technical Support 0 0 7 Documents on Demand 0 0c cece eee eee eee eee eee 10 To Reach Us by E Mail eek Ree deeds 11 Regional Offices Sales and Service 000000000 11 Blood Collection and DNA Sample Preparation 14 Proced te 4 i auos dt RA uL CLERO US P CUR RUP RAGES BA CR E 14 Low resolution SSP PCR DRB Typing 2 00 ce eee eee eee 16 OVEIVIEW 23 eee ec DER TER een MURR eee Cadet ee We ES Hg 16 Low resolution SSP PCR 0 0 0 eee eee eee nee 16 Thermal Cycling Conditions 000000002 e ee 19 Analysis of Low resolution PCR Amplifications 000 20 PEOLO CO Loa RR QR CR oS Ne CU RUP ORE e HE a 20 Low resolution Results as Control Reactions 22 Forward and Reverse Sequencing 0 0c eee eee eee eee 23 OVeEVIeW i aos o oC URL ACE CIO RT RAUS EORR a a a ioe aT ee OS QUA 23 Performing Sequencing Reactions 02 ee eee eee 23 Purifying Sequencing Reactions 0 0 0 eee eee eee ee 24 Precipitating Sequencing Reactions in a MicroAmp 9600 Tray 25 Precipitating Sequencing Reactions in Single Tubes 26 Spin Column Purification 0 0 0 0 eee eee ee eee 27 Rehydrating the Column 0 0 cece eee
24. cing reactions Step Action 1 Dilute PCR product 1 3 in DNA Diluent For sufficient sample to sequence with both codon 86 mixes add 2 uL of PCR product to 6 uL of DNA Dilution Buffer 2 To separate tubes plate wells add 8 uL of each of the codon 86 specific Ready Reaction Mixes 3 To each of the Ready Reaction Mixes in the tubes plate wells add 2 uL of diluted PCR product 4 Seal the tubes with MicroAmp caps or cover the plate with a MicroAmp 2400 or 9600 Full Plate Cover 5 Centrifuge briefly in a benchtop centrifuge to combine the PCR product and Ready Reaction Mix at the bottom of the tubes plate wells 6 Place the tubes plate in a thermal cycler 29 To perform the sequencing reactions continued Step Action 7 Do 1 cycle of the following Rapid thermal ramp to 96 C 96 C for 10 seconds 8 Repeat the following for 20 cycles 96 C for 10 seconds Rapid thermal ramp to 50 C 50 C for 10 seconds Rapid thermal ramp to 60 C 60 C for 2 minutes 9 9 9 9 9 Rapid thermal ramp to 4 C and hold until ready to precipitate the sequencing reactions To purify the samples follow the ethanol precipitation protocol described in Precipitating Sequencing Reactions in a MicroAmp 9600 Tray on page 25 or Precipitating Sequencing Reactions in Single Tubes on page 26 or Spin Column Purification on page 27 Recommendations for ABI PRISM 3
25. cludes BioLIMS BioMerge and Press FAX SQL GT applications 25 505 982 7690 PCR and Sequence press FAX Detection 5 or call 240 453 4613 1 800 762 4001 and press 1 for PCR or 2 for Sequence Detection FMAT Telephone FAX 1 800 899 5858 and press 1 then press 6 508 383 7855 Peptide and Organic f Press FAX Synthesis 31 650 638 5981 Protein Sequencing Press FAX 32 650 638 5981 Chemiluminescence Telephone FAX 1 800 542 2369 781 275 8581 U S only or Tropix 1 781 271 0045 9 00 a m to Tropix 5 00 p m ET For Support On This Product Dial 1 800 831 6844 and LC MS Telephone FAX 1 800 952 4716 650 638 6223 9 00 a m to 5 00 p m PT 10 Documents on Free 24 hour access to Applied Biosystems technical documents including MSDSs is available by fax or e mail You can access Documents on Demand through the internet or by telephone If you want to order Then United States or Canada through the Use http www appliedbiosystems com techsupport Internet You can search for documents to order using keywords Up to five documents can be faxed or e mailed to you by title by phone from the a Call 1 800 487 6809 from a touch tone phone Have your fax number ready b Press 1 to order an index of available documents and have it faxed to you Each document in t
26. e column The sample is in the sample collection tube Dry the sample in a vacuum centrifuge no heat for 10 15 minutes or until dry Do not over dry Performing Codon 86 Group Specific Sequencing Overview Performing Sequencing Reactions In some heterozygote analyses it is possible for multiple pairs of different alleles to generate the same final sequence ladder For example the heterozygous sequence from a DRB1 1102 1305 combination is identical to that from a DRB1 11041 1302 allele combination This situation is generically referred to as an ambiguous heterozygote allele combination In a majority of these cases the allele pairs are heterozygous at codon 86 Through the use of codon 86 specific sequencing primers P N 4305023 the ambiguity can be resolved The heterozygote PCR product generated in the low resolution SSP PCR is used as a Sequencing template in two separate sequencing reactions one reaction uses a sequencing primer specific for the GTG motif of codon 86 and the other reaction uses a sequencing primer specific for the GGT motif Homozygous sequences are generated in each reaction thus separating the two alleles allowing definitive allele assignment of each individual allele Note Use MicroAmp Reaction Tubes or a MicroAmp Optical 96 Well Reaction Plate Note Ensure the thermal cycling is performed with the correct method and the sample volume is set to 10 uL To perform the sequen
27. eee eee 27 Using the Column 5s eA I PAN EREBLERA S HERES 28 Performing Codon 86 Group Specific Sequencing 29 OVerVIeW Rec EA r ecg uetus iue ce Dei epu esu uns cem oet 29 Performing Sequencing Reactions cece ee eee 29 Recommendations for ABI PRISM 310 Genetic Analyzer Users 31 QCUVETVICWE vs bailar esie a e S AER ERU ea Sen be gees 31 Capillary Preparation os o sron i een 0 0 cee eee eee eee 31 Preparing and Loading Samples 00000 31 Recommendations for Sample Naming 0 33 Setting up Default Sample Names 0 02 02 33 Sample Sheet Menu choices 0 0 0 0 e eee eee eee 33 Filling in the Injection List eee 34 Signal Strengths eld oe eee oe ake ee OU Cee bee thes 34 Performing Fluorescent Signal Analysis 35 Recommendations for ABI PRISM 377 DNA Sequencer Users 36 OVeTVI W cis ete e UU e PRUNUS OE USD DADE ee RE 36 Gel Formulation 0 0 0 cece cece eh 36 Preparing and Loading Samples 0000 36 Recommendations for Sample Naming 4 37 Entering default file names for the Sample File 37 Sample Sheet Menu choices 0 000 esses 38 Loading Sample on the ABI PRISM377 sls esee 38 ABI PRISM 377 Run Parameters 0 02 c eee eee eee 39 Performing Fluorescent Signal Analysis 0 39 A
28. enters for Disease Control CDC and National Institutes of Health NIH concerning the principles of risk assessment biological containment and safe laboratory practices for activities involving clinical specimens These principles can be found in the U S Department of Health and Human Services HHS publication Biosafety in Microbiological and Biomedical Laboratories publication number 93 8395 stock number 017 040 523 7 The biosafety Level 2 containment elements are consistent with the Occupational Safety and Health Administration OSHA requirements contained in the HHS OSHA Bloodborne Pathogen Standard 29 CFR part 1910 1030 To collect and prepare blood samples Step Action 1 Collect a sample of venous blood into an ACD A EDTA or heparin tube 2 Prepare genomic DNA following appropriate blood handling procedures The following methods have been shown to yield high quality high molecular weight genomic DNA directly from samples of fresh blood QiaAmp Blood Kit Qiagen P N 29104 PureGene Gentra P N D5000 Isoquick Nucleic Acid Extraction Kit Orca P N MXT 020 100 Manual salting outa These DNA purification kits provide DNA that works well with the Applied Biosystems HLA typing kits Other methods may work but should be validated before routine use To collect and prepare blood samples continued Step Action 3 Prepare DNA dilution buffer 10 mM of Tris HC
29. es at 95 5 C 5 Vortex the tubes again then centrifuge briefly 6 Place tubes on ice until ready to load Enter sample information into the sample sheet Verify that the Sample Names you enter for the forward and reverse reactions are identical except for sequence direction as the MatchTools software searches for sequence pairs with matching Sample Names A useful format for Sample Name assignment is the specimen ID followed by the SSP PCR group a left hand square bracket and the sequence direction forward or reverse For example a sample that amplified a DRB1 G3 11 6 might have the following file names Sample ID G3 F Sample ID G3 R IMPORTANT Keep the Sample Name to 26 characters or less IMPORTANT If you do not separate the sequence direction from the Sample Name with the left hand square bracket the MatchMaker software will not correctly group the forward reverse sequences of each SSP PCR The ABI PRISM 377 Collection program allows the user to set up default file names as preferences Standardized names attached to the sample name can be created and saved Setting up default file names for a Sample File Step Action 1 Open the ABI PRISM 377 Collection software 2 Under the Window menu choose Preferences 3 Hold and release the mouse on Default File Names 37 Sample Sheet Menu choices Loading Sample on the ABI PRISM 377 Setting up default file names for a S
30. es to mix the water and gel material 4 Allow the gel to hydrate at room temperature for at least 2 hours Note Rehydrated columns can be stored for a few days at 2 6 C Longer storage in water is not recommended Allow columns that have been stored at 2 6 C to warm to room temperature before use 27 Step Action Remove any air bubbles by inverting or tapping the column and allowing the gel to settle Using the Column Do not allow any interruptions after starting the steps below Step Action 1 Remove the upper end cap first then remove the bottom cap Allow the column to drain completely by gravity Note If flow does not begin immediately apply gentle pressure to the column with a pipette bulb Insert the column into the wash tube provided Spin the column in a microcentrifuge at 730 g for 2 minutes to remove the interstitial fluid Remove the column from the wash tube and insert it into a sample collection tube e g a 1 5 mL microcentrifuge tube Remove the extension reaction mixture from its tube and load it carefully on top of the gel material Spin the column in a microcentrifuge at 730 g for 2 minutes Note If using a centrifuge with a fixed angle rotor place the column in the same orientation as it was in for the first spin This is important because the surface of the gel will be at an angle in the column after the first spin Discard th
31. g Analysis software which applies a mathematical matrix calculation using the values in the instrument file to all sample data Making a Matrix from a Sample File Run the Sequencing Standard Make Copies of the Standard Run An instrument file can be made from matrix standards or it can be made from a sample file Using a sample file requires fewer steps than running matrix standards however the matrix made from a sample file may be different from one made from matrix standards The quality of a matrix file made from a sample file depends on the quality of the sample file used This section describes making a matrix using a BigDye Terminator sequencing standard or a BigDye Terminator sequencing data file Such an instrument file containing all three matrices can be used with either dRhodamine or BigDye chemistries For more information on creating a matrix using matrix standards refer to the ABI PRISM dRhodamine Matrix Standards User Bulletin P N 904917 If problems arise such as spectral overlap in the analyzed data a new matrix can be made from any terminator sequencing sample by following the instructions below Make one sample of the BigDye Terminator Cycle Sequencing Standard P N 4304154 following the instructions on the product insert If using a sequencing data file follow the steps below substituting the appropriate file name Make three duplicate copies of the BigDye terminator standard run before creating
32. g the instruments refer to the appropriate instrument user s manual IMPORTANT This kit is not designed for use with the ABI 373 DNA Sequencer or the ABI 373 DNA Sequencer with the XL Upgrade Materials and Equipment Reagents Included The HLA DRB BigDye Terminator Sequencing Based Typing Kit Reagents and Equipment Not Included includes the reagents listed below One kit is sufficient for generating complete HLA DRB typings for 48 purified DNA samples HLA DRB Amplification Module for amplification of 48 purified DNAs One tube of AmpliTaq Gold DNA Polymerase 50 uL 5 U uL 12tubes of PCR Mixes one for each SSP PCR HLA DRB Sequencing Module sufficient for sequencing 96 templates generated with the HLA DRB Amplification Module Two tubes of HLA DRB Forward Sequencing mix Two tubes of HLA DRB Reverse Sequencing mix Two tubes of DNA Diluent Buffer In addition to the reagents supplied in this kit other items are required depending on which sequencing instrument is used Refer to the individual instrument manuals for the specific items needed Many of the items listed are available from major laboratory suppliers MLS unless otherwise noted Equivalent sources may be acceptable where noted Control and Loading Buffer Module The Control and Loading Buffer Module P N M0028 is sold separately and contains the following Two tubes of human DNA male DRB1 0301 1001 DRB3 0202 10 ng uL
33. gainst all of the alleles in the library selected in the Batch Worksheet MatchTools software then prepares a preliminary Batch Report listing the DRB allele type s that best fit s each consensus sequence Basecalls that differ within each pair of sequences Basecalls that display mismatches at constant positions or polymorphic positions for all alleles in the library Any insertions or deletions Note AX Atthe 3 end of the sequences the reverse direction does not extend as far as the forward sequence The forward sequence continues to position 270 whereas the reverse sequence is compared against the library only to position 262 41 42 HLA DRB Allele HLA DRB Allele Libraries to use for each low resolution amplification Libraries group gene Using MTNavigator Software for Editing Allele Groups Library Name Codons Included DR1 DR2 DR3 11 6 DR4 DRB1 L155 14 90 DR8 12 DR7 DR9 DR79 L2 14 78 DR10 DR10 L1 30 88 DRB3 DRB5 DRB35 L17 14 90 DRB4 DRB4 L3 28 88 For manual editing of the sequence data use MTNavigator Software to Import the Batch Worksheet containing all data files associated with a given sample The files are typically imported as a Forward Reverse pair for each SSP allele group or gene sequenced The sequences are offset such that the nucleotide numbering corresponds to the cDNA sequences as published in the nomenclature reports i e t
34. gal forces IMPORTANT Do not refrigerate during the centrifugation run IMPORTANT Be sure that these speeds are within the manufacturer s recommendations for the centrifuge 5 Immediately invert the tray on paper towels Place the inverted tray and paper towel in the centrifuge for 1 minute at 150 gto remove the supernatant IMPORTANT This centrifugation step is important to ensure complete removal of dye degradation products 6 Add 50 uL of 70 ethanol to the tubes Centrifuge for 5 minutes at 1500 3000 g 7 Immediately repeat step 5 above 25 Precipitating Sequencing Reactions in Single Tubes If there are only a few samples or you do not have access to a centrifuge with a 96 well tray adapter then a single tube ethanol precipitation procedure may be used Step Action 1 Use a 1 5 mL Microcentrifuge tube for each sequencing reaction 2 Prepare a fresh mix 1 0 mL anhydrous ethanol EtOH 40 uL 3M sodium acetate NaOAc pH 4 6 3 To each 1 5 mL microcentrifuge tube add 25 uL of the EtOH NaOAc mix 4 Pipet the entire contents of one extension reaction into one tube of EtOH NaOAc mix Mix thoroughly by vortexing 5 Spin the tubes in a microcentrifuge for 15 30 minutes at maximum speed IMPORTANT Do not refrigerate during the centrifugation run 6 Carefully aspirate the supernatant with a pipette and discard 7 Rinse the pellet with 50 uL of 7096 ethanol 8 Spin for 5 minutes in a microcentrif
35. he index has an ID number Use this as your order number in step d below c Call 1 800 487 6809 from a touch tone phone a second time d Press 2 to order up to five documents and have them faxed to you by phone from outside the United States or Canada a Dial your international access code then 1 858 712 0317 from a touch tone phone Have your complete fax number and country code ready 011 precedes the country code b Press 1 to order an index of available documents and have it faxed to you Each document in the index has an ID number Use this as your order number in step d below c Call 1 858 712 0317 from a touch tone phone a second time d Press 2 to order up to five documents and have them faxed to you Regional Offices Sales and Service your local Applied Biosystems service representative To Reach Us by Contact technical support by e mail for help in the following product E Mail areas For this product area Use this e mail address Chemiluminescence info appliedbiosystems com Genetic Analysis galab appliedbiosystems com LC MS apisupport sciex com PCR and Sequence Detection pcrlab appliedbiosystems com Protein Sequencing Peptide and DNA Synthesis corelab appliedbiosystems com If you are outside the United States and Canada you should contact The Americas United States Applied Biosystems 850 Lincoln Centre Drive Fo
36. he first position is nucleotide 40 in the cDNA sequence The first 39 bases are either in exon 1 or from the first hypervariable region of exon 2 and therefore not part of the allele library Generate shadow sequences for each pair of sequences to simplify the comparison of basecalling differences between each pair of sequences Basecall differences between the forward and reverse sequences can be due to a variety of sources including mobility anomalies or base incorporation anomalies for heterozygote positions i e one base in a heterozygote pair may not be detected at a sufficient level to be identified using the threshold value set in the Batch Worksheet See Table 3 on page 49 for a list of all currently known reproducible anomalies Perform manual editing of the sequences to resolve basecalling differences between the two orientations When the editing is complete save the changes to the sample files use the Save to files command in the Sequences drop down menu Using MatchTools During a final analysis use the MatchTools software to Software for Final Return to the preliminary analysis Batch Worksheet pales Generate a Final Batch Report by submitting the Batch Worksheet to the MatchTools software This report includes the HLA DRB allele types that best match the edited sequences Basecalls that display mismatches at constant positions or polymorphic positions for all alleles in the library 43 Ap
37. hese rights under the up front fee component may be purchased from Applied Biosystems or obtained by purchasing an Authorized Thermal Cycler Further information on purchasing licenses to practice the PCR Process may be obtained by contacting the Director of Licensing at Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 or at Roche Molecular Systems Inc 1145 Atlantic Avenue Alameda California 94501 No general patent or other license of any kind other than this specific right of use from purchase is granted hereby Notice to Purchaser About Limited License This kit reagent is sold pursuant to a limited sublicense from Amersham International plc under one or more U S Patent Nos 5 498 523 4 994 372 U S Patent Application Serial Nos 08 324437 08 337615 and corresponding foreign patents and patent applications The purchase of this kit reagent includes a limited non exclusive sublicense without the right to resell repackage or further sublicense under such patent rights to use this reagent for DNA sequencing or fragment length analysis solely with an Applied Biosystems commercial automated sequencing machine or other authorized DNA sequencing machines that have been authorized for such use by Applied Biosystems Division of the PE Corporation or for manual DNA sequencing No license is hereby granted for the use of this kit or the reagents therein in any other automated sequencing machine Such sublicense is granted
38. icular fluorescent dye Each of these areas on the CCD camera is referred to as a virtual filter since no physical filtering hardware like band pass glass filters is used The information that specifies the appropriate virtual filter settings for a particular set of fluorescent dyes is contained in each appropriate run module In the dRhodamine dyes dR110 emits at the shortest wavelength and is detected as blue followed by dR6G green dTAMRA yellow and dROX red Although each of these dyes emits its maximum fluorescence at a different wavelength there is some overlap in the emission spectra among the four dyes The goal of multicomponent analysis is to isolate the signal from each dye so that there is as little noise in the data as possible The precise spectral overlap between the four dyes is measured by running DNA fragments labeled with each of the dyes in separate lanes of a gel or in separate injections on a capillary These dye labeled DNA fragments are called matrix standard samples The Data Utility software then analyzes the data from each of these four samples and creates an instrument matrix file The instrument file contains tables of numbers with four columns and four rows These numbers are normalized fluorescence intensities and represent a mathematical description of the spectral overlap that is observed between the four dyes Multicomponent analysis of sequencing data is performed automatically by the Sequencin
39. ing A Reverse C E g A G base 155 codon 52 anomalous A mobility 162 54 Reverse All constant G S low G with high Complement position C background Reverse B B JN G B base 162 codon 54 anomalous C mobility 51 Table 3 Anomalies continued Heterozygote Sequence Combination Correct InitialBase Nature of Position Codon Orientation 1st Allele 2nd Allele Base Call Call Anomaly 169 170 57 Forward 1302 1303 RA RR low G incorporation at position 170 169 170 57 Reverse 1302 1303 AR RR low G Complement incorporation at position 169 Forward E T R r 4 8 base 170 codon 57 low G jt Reverse t T r R i y 6 base 169 codon 57 low G 52 Table 3 Anomalies continued Heterozygote Sequence Combination Correct Initial Base Nature of Position Codon Orientationa 1st Allele 2nd Allele Base Call Call Anomaly 170 171 57 Forward DRB3 DRB3 WY WT low C at position 0101 0202 Hi 170 171 57 Reverse DRB3 DRB3 WY AY low C Complement incorporation at TIO vade position 170 Forward T G wW y G base 171 codon 57 low C Reverse T 6 w Y G F base 170 codon 57 low T Table 3 Anomalies continued Heterozygote ination Sequence Combination Correct In
40. itialBase Nature of Position Codon Orientationa 1stAllele 2nd Allele Base Call Call Anomaly 201 67 Forward All constant C Y extra T shoulder position from following T Forward t T c c L B bases 201 202 codon 67 68 anomalous T mobility 207 212 69 71 Forward 1301 1401 RSA SRR RSA CAA low G at positions 210 211 212 207 212 69 71 Reverse 1301 1401 RSA SRR RCA CAA low G at Complement positions 208 210 211 212 Forward G R E i S r r G C bases 210 211 212 codons 70 71 low Qs B v a Reverse A R s A r r 8 bases 208 210 211 212 codons 70 71 low G s 54 Table 3 Anomalies continued base 217 codon 73 T background noise Heterozygote Sequence Combination Correct Initial Base Nature of Position Codon Orientationa 1st Allele 2nd Allele Base Call Call Anomaly 212 71 Forward 1301 1303 R A low G at position 212 j Forward C C S y G G base 221 codon74 low C 217 73 Forward 0301 or G K low G with T DRBS alleles background noise ee l Forward E b g 5 55 Table 3 Anomalies continued Heterozygote Sequence Combination Correct InitialBase Nature of Position Codon Orientation 1st Allele 2nd Allele Base Call Call Anomaly 220 74 Reverse 0403 G
41. mplement refers to the reverse sequences after having been processed by MatchTools The bases described are those that will be observed in MTNavigator during the editing process b Due to the large number of possible heterozygote allele combinations the alleles listed in this table are for reference only For any given sample it may be helpful to review the published allele sequences to determine whether the same motif combinations are present in the sequences being reviewed 58 IUB Codes The code A Adenosine R Aor G puRine C Oytidine Y C or T pYrimidine G Guanosine K Gor T Keto T Thymidine M A or C aMino B C GorT S Gor C Strong 3H bond D A G or T W Aor T Weak 2H bond H A C or T N aNy base V A CorG 59 Appendix D Instrument Matrix Files Multicomponent Multicomponent analysis is the process that separates the four different Analysis fluorescent dye colors into distinct spectral components The four dyes used in the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit are dR110 dR6G dTAMRA and dROX Each of these fluorescent dyes emits its maximum fluorescence at a different wavelength During data collection on the ABI PRISM 310 and 377 instruments the collection software collects light intensities from four specific areas on the CCD camera each area corresponding to the emission wavelength of a part
42. not have access to a bench top centrifuge fitted with a 96 well tray adaptor two alternative methods can be used a single tube ethanol precipitation method or a Centri Sep M spin column procedure Precipitating Sequencing Reactions in a MicroAmp 9600 Tray Perform this step in the same tubes plate wells as the cycle sequencing reaction This helps minimize the number of sample transfers and potential sample mix ups Note Note Use the MicroAmp 9600 Base and Tray Retainer to hold the tubes If multiple samples are processed in a 96 well format using a multichannel pipettor can simplify precipitation IMPORTANT order they are given If the precipitated reactions sit between steps the signal strength will be decreased IMPORTANT Remove the ethanol completely from the precipitated reactions as it may contain unincorporated terminators and dye degradation products To perform ethanol precipitation Perform the steps below without interruption and in the exact Step Action 1 Prepare a fresh mix 1 0 mL anhydrous ethanol EtOH 40 uL 3M sodium acetate NaOAc pH 4 6 To each sequencing reaction add 25 uL of the EtOH NaOAc mix Replace the strip caps or full plate cover and mix thoroughly by vortexing or inversion 4 In a centrifuge fitted with a 96 well microtiter tray adaptor centrifuge for 30 minutes at 1500 3000 g see Spin Column Purification on page 27 to calculate relative centrifu
43. nts and have them faxed or e mailed to you through our site see the Documents on Demand section below In the United States and Canada technical support is available at the following times Product Hours Chemiluminescence 9 00 a m to 5 00 p m Eastern Time LC MS 9 00 a m to 5 00 p m Pacific Time All Other Products 5 30 a m to 5 00 p m Pacific Time See the Regional Offices Sales and Service section below for how to contact local service representatives outside of the United States and Canada Call Technical Support at 1 800 831 6844 and select the appropriate option below for support on the product of your choice at any time during the call To open a service call for other support needs or in case of an emergency press 1 after dialing 1 800 831 6844 For Support On This Product Dial 1 800 831 6844 and ABI PRISM9 3700 DNA Press FAX Analyzer 8 650 638 5981 ABI PRISM 3100 Genetic Analyzer Press FAX 26 650 638 5891 DNA Synthesis Press FAX 21 650 638 5981 For Support On This Product Dial 1 800 831 6844 and Fluorescent DNA gt Press FAX Sequencing 22 650 638 5891 Fluorescent Fragment Press FAX Analysis includes GeneScan applications 23 650 638 5891 Integrated Thermal Cyclers Press FAX 24 650 638 5891 Biolnformatics in
44. or Bis Acrylamide Acrylamide and Bis Acrylamide are poisons neurotoxins irritants carcinogens and possible teratogens Acrylamide and Bis Acrylamide sublime the solid releases toxic vapor and are harmful if swallowed inhaled or absorbed through the skin Effects are cumulative When handling always wear personal protective equipment i e lab coat safety glasses and chemical resistant gloves and use in a well ventilated area Thoroughly clean surfaces subject to gel contamination i e binder clips combs bench tops etc IMPORTANT To ensure adequate signal strength perform the steps below in the exact order they are given Preparing the Samples Step Action 1 Combine recrystallized formamide and blue dextran EDTA solution in a 5 1 ratio to prepare the sequencing loading buffer Make fresh for each use WARNING CHEMICAL HAZARD Formamide is a known teratogen It can cause birth defects Obtain a copy of the MSDS from Applied Biosystems Web site or Fax on Demand see p 8 Wear appropriate protective eyewear clothing and gloves Wash thoroughly after handling formamide Recommendations for Sample Naming Entering default file names for the Sample File Step Action 2 Resuspend each DNA pellet in 4uL of sequencing loading buffer 3 Vortex the tubes then centrifuge briefly 4 Denature each sample by placing in a heating block or thermal cycler for 2 minut
45. owing Autoanalysis it may be necessary to process the data files Choose which data files to analyze then analyze and print them using the Sequencing Analysis software Choosing Files for Analysis Step Action 1 Launch the Sequencing Analysis software The Sample Manager window Sample File Queue in versions 2 1 1 and 2 1 2 appears 2 Click the Add Files button A dialog box appears 3 Double click each file to be analyzed then click Done 4 Choose the ABI 100 basecaller for each file 39 Generating DNA Sequences from Sample Files Step Action 1 Set the Start Point and Stop Point using the following procedure a Double click on the first file to be analyzed and view the raw data b Starting at the beginning of the raw data file scroll along the data by clicking and holding the right direction arrow at the bottom of the dialog box c Continue scrolling until the cursor is beyond the broad fluorescent peaks at the beginning of the sequencing ladder d Set the Start Point to the right of the last broad fluorescent peak e Set the Stop Point to be at the end of the sequence run If too many bases are processed the excess sequence will make it difficult for the MatchMaker software to define the sequence orientation Version 3 0 users may want to set the endpoint in the Basecaller settings to 8 Ns in 10 Be aware that any sequence region containing 8 Ns in 10 bases will be
46. pendix A HLA DRB Typing Results Template Sample Form Use a form like the template below to indicate positive and negative low resolution grouping results for each sample Sample Name DRB1 G1 DRB1 G2 DRB1 G3 11 6 DRB1 G4 DRB1 G7 DRB1 G8 12 DRB1 G9 DRB1 G10 DRB3 DRB4 DRB5 DRB ALL PCR Information Thermal Cycler Date of Gel Operator Sequencing Cross reference Information Sequencer Date Folder Name Comments Attach Photograph of Agarose Gel Appendix B HLA DRB Comparison Table Low Resolution SSP PCR Results The following table presents the normal low resolution SSP PCR expected results These relationships are based on the normal haplotype linkage patterns between DRB1 and the other three expressed DRB genes DRB1 DRB1 DRB3 DRB4 DRB5 DRB1 DRB1 DRB3 DRB4 DRB5 1 x 3 11 6 4 2 E 3 11 6 7 3 11 6 7 3 11 6 8 12 8 4 3 11 6 8 12 12 7 3 11 6 9 x 8 12 8 3 11 6 10 8 12 12 m x 4 7 9 x 7 4 8 12 8 10 4 8 12 12 1 2 4 9 m 1 3 11 6 4 10 1 4 7 8 12 8 7 1 7 7 8 12 12 1 8 12 8 T
47. plied in the HLA Control and Loading Buffer Module Over time formamide decomposes to formate Formate ions are injected preferentially into the capillary causing a loss of signal intensity 2 Vortex the tubes then centrifuge briefly 3 Denature samples by placing the tray in a heating block or thermal cycler for 2 minutes at 95 5 C 4 Vortex the tubes again then centrifuge briefly 5 Seal each tube securely using the strip septa 6 Secure the strip septa with the retainer clips 31 32 To prepare and load the samples from a MicroAmp 9600 tray Step Action 7 Place the sample tray on the autosampler IMPORTANT Do not let the denatured samples sit for more than 48 hours before injection To prepare and load the samples in 0 5 mL Genetic Analyzer Sample Tubes Step Action 1 Resuspend the DNA pellets in 15 uL of the recrystallized formamide provided in the HLA Control and Loading Buffer Module WARNING CHEMICAL HAZARD Formamide is a known teratogen It can cause birth defects Obtain a copy of the MSDS from Applied Biosystems webpage or Fax on Demand see p 8 Wear appropriate protective eyewear clothing and gloves Wash thoroughly after handling formamide IMPORTANT Use only the recrystallized formamide supplied in the HLA Control and Loading Buffer Module Over time formamide decomposes to formate Formate ions are injected preferen
48. precipitate and denature referring to details in Preparing and Loading Samples on page 31 for the ABI PRISM 310 DNA Sequencer or Preparing and Loading Samples on page 36 for the ABI PRISM 377 DNA Sequencer Source of ethanol Check reagent quality Confirm that the ethanol is absolute or anhydrous Noisy baseline Weak signal less than 50 fluorescent units See Weak signal strengths details above Signal too strong Note Ifthe signal is too strong multicomponent analysis will not be performed properly Dilute the sequence ladder and re load For the ABI PRISM 310 user decrease injection time Incorrect run module filter set Repeat the run using the correct module Run Module HLA DRB BDT Poor matrix Make a new matrix See Appendix D on page 60 Broad fluorescent terminator artifacts dye blobs Failure to perform 70 ethanol wash Re sequence and precipitate referring to details in Purifying Sequencing Reactions on page 24 Failure to completely remove supernatant following anhydrous ethanol precipitation Re sequence and precipitate referring to details in Precipitating Sequencing Reactions in a MicroAmp 9600 Tray on page 25 or Precipitating Sequencing Reactions in Single Tubes on page 26 Note For more general sequencing problems e g poor resolution base spacing etc refer to the AB
49. r continued Item Source For the ABI PRISM 310 Genetic Analyzer Only ABI PRISM 310 Genetic Analyzer capillary Applied Biosystems labeled with a green mark L 47 cm P N 402839 Lg 36 cm i d 50 um ABI PRISM 310 Genetic Analyzer Sample Tubes Applied Biosystems 0 5 mL P N 401957 ABI PRISM 310 Genetic Analyzer septa for Applied Biosystems 0 5 mL sample tubes P N 401956 ABI PRISM 310 Genetic Analyzer Buffer Vials Applied Biosystems 4 0 mL P N 401955 Genetic Analyzer Retainer Clips Applied Biosystems P N 402866 Genetic Analyzer Septa Strips 0 2 mL Tube Applied Biosystems P N 402059 Glass syringe 1 0 mL Applied Biosystems P N 604418 Safety Information For information on the safe operation of the ABI PRISM 310 Genetic Analyzer refer to the ABI PRISM 310 Genetic Analyzer Site Preparation and Safety Guide For information on the safe operation of the ABI PRISM 377 DNA Sequencer refer to the ABI PRISM 377 DNA Sequencer Safety Summary Technical Support To Reach Us on the Web Hours for Telephone Technical Support To Reach Us by Telephone or Fax in North America Applied Biosystems web site address is http www appliedbiosystems com techsupport We strongly encourage you to visit our web site for answers to frequently asked questions and to learn more about our products You can also order technical documents and or an index of available docume
50. rds for Histocompatibility Testing American Society for Histocompatibility and Immunogenetics 1996 Membership Directory pp 17 28 Sequencing SSP PCR Fragments Allele Assignment Instrument Platforms The positive amplification reactions from the SSP PCR are used as sequencing templates to generate the high resolution allele typing information The PCR and sequencing protocols enable direct sequencing with no PCR purification steps The design of the amplification primers allows sequencing of both strands from a single amplification fragment The BigDye terminator cycle sequencing chemistry provided in the Ready Reaction format has been optimized for ease of use and high throughput with less technician time The final step in the analysis procedure is to perform the allele assignment using MatchTools Software and MTNavigator Software provided in the MatchMaker Allele Identification Software package These programs work together to assign alleles and to allow manual review or editing of the sequencing data The protocols provided in this document were optimized using the GeneAmp PCR System 2400 and GeneAmp PCR System 9600 Thermal Cyclers Specific instructions are given for the HLA DRB kit reagents to generate data on the ABI PRISM 310 Genetic Analyzer and the ABI Prism 377 DNA Sequencer The protocol identifies certain steps where the protocol varies between the instruments For more detailed instructions on usin
51. sample file used to make the matrix 3 Select the new matrix under Instrument File 4 Reanalyze the data file 5 Look at the Analyzed data window Peaks of only one color should be seen Figure 1 Peaks under peaks indicate a poor or incorrect matrix Figure 2 6 If the sequence quality is poor do one of the following a Modify the Make Matrix default values b See Note on preceding page PETI EE COGS CORO SINORT DE CET DIRE CARER G2 Pe CRT ORC DI Ce UG E ECA TEAG GAR mem Figure 1 Correct matrix ppm op CIE MIGG O6 DE E CRUCE SUD R ee ee ee oR ORE TERRITORIO OT S TCATCGACGGCA 120 30 140 150 170 180 Figure 2 Incorrect matrix Worldwide Sales Offices Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches into 150 countries on six continents For international office locations please call our local office or refer to our web site at www appliedbiosystems com Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free 1 800 345 5224 Fax 1 650 638 5884 Technical Support For technical support Toll Free 1 800 831 6844 ext 23 Fax 1 650 638 5891 www appliedbiosystems com LI AR Applied sw Biosystems Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera Corpor
52. ssignment of HLA DRB Allele Types to Sequence Files 41 OVERVIEW v veu eere reote meo eges SE bd Heke 41 Using MatchTools Software for Preliminary Analysis 41 HLA DRB Allele Libraries 0 0 0 0 0 een 42 Using MTNavigator Software for Editing 42 Using MatchTools Software for Final Analysis 43 Appendix A HLA DRB Typing Results Template 44 Sample FOr soceri GA CENT RON ER RR EE 44 Appendix B HLA DRB Comparison Table 0 0 45 Low Resolution SSP PCR Results 0000000002 45 Appendix C Troubleshooting Guide 00 00 0000 46 OVerVIe We oU eR a C LR RIAL BON ERR UR RACE EAR 46 POR R5 e tide site eb utuostut ees 46 SEQUENCING dose deae We SALUS WIS 46 MatchTools Software 20 0c eee eee eee nee 48 Anomalies Table 0 0 0 cee cc cee eee eens 49 IUB COd s 1d bea UR Mae thee Oat eh Be Lewes ES eS 59 Appendix D Instrument Matrix Files 0 00 eese 60 Multicomponent Analysis ee eee 60 Making a Matrix from a Sample File 0000 61 Run the Sequencing Standard 00 0000 02 2 61 Make Copies of the Standard Run 0 0 0000 61 View Matrix Run Raw Data 61 Generate the Terminator Matrix ssl 61 Complete the Other Matrices llle 63 Evaluate Matrix Quality 0 0 0 eee eee 64 Introduction Overview SSP
53. ster City California 94404 Tel 650 570 6667 800 345 5224 Fax 650 572 2743 Latin America Del A Obregon Mexico Tel 305 670 4350 Fax 305 670 4349 Europe Austria Wien Hungary Budapest Tel 43 0 1 867 35 750 Tel 36 0 1 270 8398 Fax 43 0 1 867 35 75 11 Fax 36 0 1 270 8288 Belgium Tel 32 0 2 712 5555 Fax 32 0 2 712 5516 Italy Milano Tel 39 0 39 83891 Fax 39 0 39 838 9492 Czech Republic and Slovakia Praha Tel 420 2 61 222 164 Fax 420 2 61 222 168 The Netherlands Nieuwerkerk a d IJssel Tel 31 0 180 331400 Fax 31 0 180 331409 Denmark Naerum Tel 45 45 58 60 00 Fax 45 45 58 60 01 Norway Oslo Tel 47 23 12 06 05 Fax 47 23 12 05 75 11 12 Europe Finland Espoo Tel 358 0 9 251 24 250 Fax 358 0 9 251 24 243 Poland Lithuania Latvia and Estonia Warszawa Tel 48 22 866 40 10 Fax 48 22 866 40 20 France Paris Tel 33 0 1 69 59 85 85 Fax 33 0 1 69 59 85 00 Portugal Lisboa Tel 351 0 22 605 33 14 Fax 351 0 22 605 33 15 Germany Weiterstadt Tel 49 0 6150 1010 Fax 49 0 6150 101 101 Russia Moskva Tel 7 095 935 8888 Fax 7 095 564 8787 Spain Tres Cantos Tel 34 0 91 806 1210 Fax 34 0 91 806 1206 South Africa Johannesburg Tel 27 11 478 0411 Fax 27 11 478 0349 Sweden Stockholm Tel 46 0 8 619 4400 Fa
54. t Allele 2nd Allele Base Call Call Anomaly 47 16 Forward All constant CA CM mobility shift position Forward 6 T C a T T T base 47 codon 16 anomalous C mobility 100 101 34 Forward All constant position CA MM mobility shift 1 4 E c a G Forward base 100 101 codon 34 anomalous C A mobility 49 Table 3 Anomalies continued Heterozygote Sequence Combination Correct InitialBase Nature of Position Codon Orientation2 1st Allele 2nd Allele Base Call Call Anomaly 112 113 38 Forward 1301 1401 SY GY low C incorporation 112 113 38 Reverse 1301 1401 SY GY low C Complement incorporation Forward M i i 5 i base 112 codon 38 low C Reverse 2 n w E E Y G c base 112 codon 38 low C 136 46 Reverse All constant G R extra A shoulder Complement position from following A Reverse 8 8 g x 8 T base 136 codon 46 anomalous A mobility nd Vv 50 Table 3 Anomalies continued Heterozygote ination Sequence Combination Correct Initial Base Nature of Position Codon Orientation ist Allele 2nd Allele Base Call Call Anomaly 155 52 Reverse All constant G R extra A shoulder Complement position from follow
55. tially into the capillary causing a loss of signal intensity Vortex the tubes then centrifuge briefly Denature each sample by placing in a heating block or thermal cycler for 2 minutes at 95 5 C 4 Vortex the tubes again then centrifuge briefly 5 Transfer the samples into 0 5 mL Genetic Analyzer Sample Tubes Label one tube for each sample IMPORTANT Do not transfer to 0 5 mL tubes before the sequences have been heat denatured This may lead to lower or inadequate signal strengths 6 Seal each tube with a septum Press the septa onto the tubes with a slight twist of the thumb 7 Place the tubes in the 48 well sample tray 8 Place the sample tray on the autosampler IMPORTANT Do not let the denatured samples sit for more than 48 hours before injection Recommendations for Sample Naming Setting up Default Sample Names Sample Sheet Menu choices Enter sample information into the sample sheet Verify that the Sample Names you enter for the forward and reverse reactions are identical except for sequence direction as the MatchTools software searches for sequence pairs with matching Sample Names A useful format for Sample Name assignment is the specimen ID followed by the SSP PCR group a left hand square bracket and the sequence direction forward or reverse For example a sample that amplified a DRB1 G3 11 6 might have the following file names Sample ID G3 F Sample ID G3 R IMPORTANT
56. to 99 C 99 C for 10 min Do 1 cycle of the following Rapid thermal ramp to 4 C 4 forever After amplification store PCR products at 2 6 C until needed 19 Analysis of Low resolution PCR Amplifications Protocol To analyze low resolution PCR amplifications Step Action 1 Prepare a 2 agarose gel containing 1X TBE buffer and 0 5 ug mL ethidium bromide WARNING CHEMICAL HAZARD Ethidium bromide is a known mutagen It can change genetic material in a living cell Before using ethidium bromide as a stain read the manufacturer s MSDS which gives information on physical characteristics hazards precautions first aid spill clean up and disposal procedures Wear appropriate protective eyewear clothing and gloves Combine 7 5 uL of each PCR amplification reaction with 3 uL of agarose gel loading buffer Load 10 uL on the agarose gel Load in one lane 0 5 ug of BRL 100 bp or 250 bp size ladder in loading buffer Run the gel in 1X TBE at 10 V cm until the fragments in the size ladder resolve For example a 100 mL 10 5 cm submarine gel run at 150 V for 30 minutes is sufficient To analyze low resolution PCR amplifications continued Step Action 6 Photograph the resulting electrophoresis pattern The photograph below shows that for this sample the DRB1G4 DRB1G7 and DRB4 alleles are present DRB1G3 11 6 DRB1G2 DRB1G4 D
57. uge at maximum speed Again carefully aspirate the supernatant and discard 9 Dry the pellet in a vacuum centrifuge Do not over dry Spin Column We recommend Centri Sep spin columns from Princeton Separations P N CS 901 This protocol is different from earlier spin column protocols The following changes have been made Purification Rehydrating the Column Do not spin the column at speeds 2735 g see formula below Do not spin more than 2 minutes The rehydration time has been increased to 2 hours Tips for optimizing spin column purification Use one column for each sample Do not process more columns than you can handle conveniently at one time Load the sample in the center of the column bed without touching the resin Make sure that the sample does not touch the sides of the column Spin the column at 325 730 g for best results Use the following formula to calculate the best speed for your centrifuge g 11 18 r rpm 1000 where g relative centrifugal force rpm revolutions per minute r radius of the rotor in cm The entire procedure should be performed without interruption to ensure optimal results Do not allow the column to dry out Step Action 1 Gently tap the column to cause the gel material to settle to the bottom of the column 2 Remove the upper end cap and add 0 8 mL of deionized water 3 Replace the upper end cap and invert the column a few tim
58. w corresponds to a different sample The first eleven columns in each row contain Allele Group specific PCR Mix genomic template and AmpliTaq Gold DNA Polymerase The twelfth column contains DRB ALL PCR Mix as either a positive or negative control 17 To perform low resolution SSP PCR continued Step Action DRB1G2 DRB ALL Samples G UO OOO OOOO GOO O Q prea O00000 OOOO OO Q orB1483 11 6 000000 e msc 000 OO Qv mec OOOO OO e ms srz OOOO OO Q bs OOOO OO Q ns OOOO QO 9Q w s O OO QO Q O pores OOOO O00 prs O O O VY VY Ve 3 Spin tubes briefly in a tabletop centrifuge fitted with a 96 well or 24 well tray support to collect their contents at the bottoms of the tubes 4 Seal the tubes with MicroAmp Caps or cover them with a MicroAmp 9600 or 2400 Full Plate Cover 5 Place the tray in the thermal cycler GeneAmp PCR System 9600 or 2400 Thermal Cycling For either the GeneAmp PCR System 9600 or 2400 set the reaction Conditions volume to 12 uL Program the thermal cycler to Step Action 1 Do 1 cycle of the following to activate AmpliTaq Gold Rapid thermal ramp to 95 C 95 for 10 min Repeat the following for 36 cycles 96 C for 20 seconds Rapid thermal ramp to 65 C 65 C for 30 seconds Rapid thermal ramp to 72 C 72 C for 30 seconds 9 9 9 9 Do 1 cycle of the following Rapid thermal ramp
59. x 46 0 8 619 4401 United Kingdom Warrington Cheshire Tel 44 0 1925 825650 Fax 44 0 1925 282502 Switzerland Rotkreuz Tel 44 0 41 799 7777 Fax 41 0 41 790 0676 South East Europe Zagreb Croatia Tel 385 1 34 91 927 Fax 385 1 34 91 840 Middle Eastern Countries and North Africa Monza Italia Tel 39 0 39 8389 481 Fax 39 0 39 8389 493 Africa English Speaking and West Asia Fairlands South Africa Tel 27 11 478 0411 Fax 27 11 478 0349 All Other Countries Not Listed Warrington UK Tel 44 0 1925 282481 Fax 44 0 1925 282509 Japan Japan Hatchobori Chuo Ku Tokyo Tel 81 3 5566 6100 Fax 81 3 5566 6501 Eastern Asia China Oceania Australia Scoresby Victoria Malaysia Petaling Jaya Tel 61 3 9730 8600 Tel 60 3 758 8268 Fax 61 3 9730 8799 Fax 60 3 754 9043 China Beijing Singapore Tel 86 10 6238 1156 Tel 65 896 2168 Fax 86 10 6238 1162 Fax 65 896 2147 Hong Kong Taiwan Taipei Hsien Tel 852 2756 6928 Tel 886 2 2698 3505 Fax 852 2756 6968 Fax 886 2 2698 3405 Korea Seoul Tel 82 2 593 6470 6471 Fax 82 2 593 6472 Thailand Bangkok Tel 66 2 719 6405 Fax 66 2 319 9788 13 Blood Collection and DNA Sample Preparation 14 Procedure WARNING BIOHAZARD Blood samples have the potential to transmit infectious diseases Follow the latest guidelines published by the C
Download Pdf Manuals
Related Search