instructions for use gendx sbt excellerator® · eighth edition july 2011 sample & assay...

Eighth Edition July 2011

Sample & Assay Technologies

Instructions For Use

GenDx SBTexcellerator®

For high-resolution HLA Sequencing Based Typing (SBT)

Genome Diagnostics B.V.

Phone: +31 302 523 799

Email: [email protected]

Web: www.gendx.com

Address: Yalelaan 48

3584 CM Utrecht

the Netherlands

Distributed by

0344

CE Marking

Catalog Number CE Marking

SBTexcellerator® HLA-A 4100084, 4100184 CE 0344

SBTexcellerator® HLA-B 4101084, 4101184 CE 0344

SBTexcellerator® HLA-C 4102084, 4102184 CE

SBTexcellerator® HLA-DRB1 4105084, 4105184 CE 0344

SBTexcellerator® HLA-DRB3/4/5 4105384 CE 0344

SBTexcellerator® HLA-DQA1 4106084, 4106184 n.a.

SBTexcellerator® HLA-DQB1 4104084, 4104184 CE

SBTexcellerator® HLA-DPB1 4103084, 4103184 CE

Copyright This publication, including all photographs, illustrations, is protected under international copyright laws, with all rights reserved. Neither this manual, nor any of the material contained herein, may be reproduced without written consent of the author. © Copyright 2011 Disclaimer Genome Diagnostics B.V. has made every effort to ensure that this Instructions For Use is accurate. Genome Diagnostics B.V. disclaims liability for any inaccuracies or omissions that may have occurred. Information in this Instructions For Use is subject to change without notice. Genome Diagnostics B.V. assumes no responsibility for any inaccuracies that may be contained in this Instructions For Use. Genome Diagnostics B.V. reserves the right to make improvements to this Instructions For Use and/or to the products described in this Instructions For Use, at any time without notice. If you find information in this manual that is incorrect, misleading, or incomplete, we would appreciate your comments and suggestions. Please send them to [email protected].


Contents

CE Marking 2

Contents 3

Key to Symbols 4

Kit Content 5

For HLA Class I 5

For HLA Class II 7

Shipping and Storage 9

Warning and Precautions 9

Product Use Limitations 9

Safety Information 10

Intended Use 10

Technical Assistance 10

Principle and Procedure 10

Important notes before starting 11

Sample preparation 11

SBTexcellerator® primer preparation 11

Assay set-up 11

Equipment and Reagents to be supplied by User 12

Protocol 1A: HLA Class I Loci Amplification 14

Protocol 1B HLA-DRB Loci Amplification 17

Protocol 1C: HLA-DQA1 Locus Amplification 21

Protocol 1D: HLA-DQB1 Locus Amplification 24

Protocol 1E: HLA-DPB1 Locus Amplification 27

Protocol 2: PCR product Clean-up 30

Protocol 3: Sequencing of HLA Loci 31

Protocol 4: Clean-up and Analysis of HLA Sequencing Products 33

Troubleshooting Guide 35

Appendix A: Control of Contamination 39

Notes 41

Key to Symbols

Material Number

Components

In Vitro Diagnostics Medical Device

Batch Code/ Lot Number

Catalogue Number

Consult Instructions for use

Contains reagents for N tests

Store at -20°C Store at -20°C

Legal Manufacturer

Add liquid


Kit Content

For HLA Class I

SBTexcellerator® HLA-A Kits Core Kit (50) Extd. Kit (50)

Catalog no. 4100084 4100184

Number of reactions 50 50

HLA-A Amplification Primers (red cap) 1 tube –

HLA-A Sequencing Primers (yellow cap) Multiple tubes* –

HLA-A GSSP Primers (green cap) – Multiple tubes*

Nuclease Free H2O (clear cap) 1 tube 1 tube

HLA-A Protocol Sheet 3000101 –

Instructions For Use 3840000 –

SBTexcellerator® HLA-B Kits Core Kit (50) Extd. Kit (50)

Catalog no. 4101084 4101184


HLA-B Amplification Primers (red cap) 1 tube –

HLA-B Sequencing Primers (yellow cap) Multiple tubes* Multiple tubes*

HLA-B GSSP Primers (green cap) – Multiple tubes*


HLA-B Protocol Sheet 3000201 –

Instructions for Use 3840000 –

* See the protocol sheet for primer details.

SBTexcellerator® HLA-C Kits Core Kit (50) Extd. Kit (50)

Catalog no. 4102084 4102184


HLA-C Amplification Primers (red cap) 1 tube –

HLA-C Sequencing Primers (yellow cap) Multiple tubes* Multiple tubes*

HLA-C GSSP Primers (green cap) – Multiple tubes*

Nuclease Free H2O(clear cap) 1 tube 1 tube

HLA-C Protocol Sheet 3000301 –




For HLA Class II

SBTexcellerator® HLA-DRB Kits

HLA-DRB1 Core (50)

HLA-DRB1 Extd. (50)

HLA-DRB3/4/5 (50)

Catalog no. 4105084 4105184 4105384

Number of reactions 50 50 50

HLA-DRB1 Amplification Primers (red cap)

1 tube – –


– – 1 tube


– – 1 tube


– – 1 tube

HLA-DRB1 Sequencing Primers (yellow cap)

Multiple tubes*

1 tube –

HLA-DRB3/4/5 Sequencing Primers (yellow cap)

– – Multiple tubes*

HLA-DRB1 GSSP primers (green cap)

1 tube Multiple tubes*

–

HLA-DRB3/4/5 GSSP primers (green cap)

– – 1 tube

Nuclease Free H2O

(clear cap) 1 tube 1 tube 1 tube

HLA-DRB Protocol Sheet 3000601 – 3000701

Instructions for Use 3840000 – 3840000


SBTexcellerator® HLA-DQA1 Kits Core Kit (50) Extd. Kit (50)

Catalog no. 4106084 4106184


HLA-DQA1 Amplification Primers (red cap) 2 tubes –

HLA-DQA1 Sequencing Primers (yellow cap) Multiple tubes* Multiple tubes*


HLA-DQA1 Protocol Sheet 3000801 –


SBTexcellerator® HLA-DQB1 Kits Core Kit (50) Extd. Kit (50)

Catalog no. 4104084 4104184


HLA-DQB1 Amplification Primers (red cap) 1 tube –

HLA-DQB1 Sequencing Primers (yellow cap) Multiple tubes* –

HLA-DQB1 GSSP Primers (green cap) – Multiple tubes*


HLA-DQB1 Protocol Sheet 3000501 –




SBTexcellerator® HLA-DPB1 Kits Core Kit (50) Extd. Kit (50)

Catalog no. 4103084 4103184


HLA-DPB1 Amplification Primers (red cap) 1 tube –

HLA-DPB1 Sequencing Primers (yellow cap) Multiple tubes* Multiple tubes*

HLA-DPB1 GSSP Primers (green cap) – Multiple tubes*


HLA-DPB1 Protocol Sheet 3000401 –



Shipping and Storage

SBTexcellerator® HLA Kits are:

- shipped at ambient temperature and should be stored at -20°C upon arrival

- stable until the kit expiration date when stored at -20°C

- stable for 5 months after dissolving primers in nuclease free H2O when stored at -20°C

Warning and Precautions

Product Use Limitations

To ensure the best performance, use the SBTexcellerator® HLA Kits with the materials, reagents, and equipments recommended in “Equipment and Reagents to be supplied by User”, page 12. Use of materials other than specified, must be validated by user!

Reconstitution or dilution of primers in volumes other then described in these Instructions For Use is strongly discouraged!

Please take special note of Appendix A: Control of Contamination on page 39.

Safety Information

When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles.

Intended Use

SBTexcellerator® products are intended for high-resolution identification of alleles of the Human Leukocyte Antigens (HLA) by means of Sequencing Based Typing (SBT). SBTexcellerator® products can be used for donor registry typing and transplantation diagnostics. The specimen material is human genomic DNA. SBTexcellerator® products are intended for in vitro diagnostic use by professional health care personnel, such as laboratory technicians and physicians, trained in HLA-typing and DNA sequencing in diagnostic laboratories either EFI or ASHI accredited or able to work according to EFI or ASHI specifications.

Technical Assistance

For technical assistance and more information, please see the Technical Support Center at www.qiagen.com/Support or call one of the QIAGEN Technical Service Departments or local distributors.

Principle and Procedure

SBTexcellerator® HLA Kits are primer/oligonucleotide sets dedicated for high-resolution HLA sequencing Based typing (SBT). For high resolution typing of HLA Class I and Class II, sequencing of the exons 2, 3 and 4 of the HLA locus can be performed (exon 4 sequencing not available for al loci) using the sequencing primers in the SBTexcellerator® HLA Core Kits. Sequencing of other exons or the use of group-specific sequencing primers (GSSP), included in the SBTexcellerator® HLA Extended Kits, is only required when ambiguities need to be resolved. 1. HLA locus-specific amplification is performed in a thermal cycler using the

amplification primer mix, template DNA, and the QIAGEN LongRange PCR Kit.

2. Before sequencing, the PCR products are cleaned up using Exonuclease I and Shrimp Alkaline Phosphatase (or alternative methods) to remove unincorporated primers and nucleotides.

3. Sequencing is performed using BigDye® Terminator sequencing chemistry. 4. The sequence products are purified using Sephadex G-50 Superfine (or

alternative methods) to remove unincorporated sequencing primers and residual nucleotides.

5. Denatured samples are loaded on an automated genetic analyzer.


Important notes before starting

Sample preparation

� Purified DNA should have an A260/A280 ratio between 1.7 and 1.9. If necessary, DNA should be diluted in Nuclease Free H2O before use.

� Blood samples should be collected in tubes with ACD or EDTA as an anticoagulant. Do NOT use heparinized samples.

SBTexcellerator® primer preparation

� BrBrBrBriefly centrifuge the tubes containing the amplification primers (red caps) iefly centrifuge the tubes containing the amplification primers (red caps) iefly centrifuge the tubes containing the amplification primers (red caps) iefly centrifuge the tubes containing the amplification primers (red caps) and sequencing primers (yellow and green caps) before opening for the and sequencing primers (yellow and green caps) before opening for the and sequencing primers (yellow and green caps) before opening for the and sequencing primers (yellow and green caps) before opening for the first time. Resuspend each primer in first time. Resuspend each primer in first time. Resuspend each primer in first time. Resuspend each primer in Nuclease Free HNuclease Free HNuclease Free HNuclease Free H2222OOOO (provided), using (provided), using (provided), using (provided), using the resuspenthe resuspenthe resuspenthe resuspension volumes listed in Tabsion volumes listed in Tabsion volumes listed in Tabsion volumes listed in Table 1.le 1.le 1.le 1.

Assay set-up

� Read the QIAGEN LongRange PCR Handbook, paying particular attention to the “Safety Information” and “Important Notes” sections before beginning the procedure.

� Set up all reactions on ice.

Table 1. Resuspension volumes for amplification primers and sequencing primers (resuspend each primer in the provided Nuclease Free H2O)

Loci Amplification primers

(red caps) Sequencing primers

(yellow and green caps)

HLA-A 55 µl 55 µl

HLA-B 55 µl 55 µl

HLA-C 55 µl 55 µl

HLA-DRB1 55 µl 55 µl

HLA-DRB3/4/5 55 µl 110 µl

HLA-DQA1 55 µl 110 µl

HLA-DQB1 55 µl 55 µl

HLA-DPB1 55 µl 55 µl

Equipment and Reagents to be supplied by User

When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate material safety data sheets (MSDSs), available from the product supplier.

For HLA locus amplification

� QIAGEN LongRange PCR Kit

� Ice

� Pipettes and pipette tips (use of pipette tips with hydrophobic filters is strongly recommended)

� Thermal cycler

� Micro centrifuge

� Vortexer

� PCR tubes (use thin-walled 0.2 ml PCR tubes recommended by the manufacturer of your thermal cycler)

� Agarose gel electrophoresis system

For sequencing of HLA loci

� Exonuclease I and Shrimp Alkaline Phosphatase; alternative clean-up methods can be used

� Pipettes and pipette tips

� Thermal cycler

� Micro centrifuge

� 1.5 or 2 ml micro centrifuge tubes

� BigDye Terminator v1.1 Cycle Sequencing Kit or BigDye Terminator v3.1 Cycle Sequencing Kit*

* This is not a complete list of suppliers and does not include many important vendors of biological supplies.


For clean-up and analysis of HLA sequencing products

Note:Note:Note:Note: Alternatively, other size-exclusion methods or ethanol precipitation of sequencing products can be used.

� Sephadex G-50 Superfine

� Multiscreen 45 ul Column Loader

� Multiscreen Column Loader Scraper Clear

� Multiscreen-HV

� Multiscreen Centrifuge Align Frame Blue

� EU Frosted Sub skirted Thin-wall 96 x 0.2 ml plates

� Pipettes and pipette tips

� Multichannel pipette (recommended to facilitate handling)

� Centrifuge; rotor and adapters must be capable of centrifuging 96 wells microtiter plates)

� Deionized water

� Capillary sequencer (e.g., ABI PRISM® 3100 or Applied Biosystems 3130 Genetic Analyzers, or ABI PRISM 3700 or Applied Biosystems 3730 DNA Analyzers, Applied Biosystems) §†

� SBTengine® software or other suitable HLA typing software to analyze sequence files and to create HLA typing reports

§ This is not a complete list of suppliers and does not include many important vendors of biological supplies.

† SBTexcellerator® HLA Kits have been tested with various polymers and sequencing chemistries.

Protocol 1A: HLA Class I Loci Amplification

This protocol is for amplification of HLAHLAHLAHLA----A, HLAA, HLAA, HLAA, HLA----B, or HLAB, or HLAB, or HLAB, or HLA----CCCC genes using the QIAGEN LongRange PCR Kit and the respective SBTexcellerator® HLA Kit.

Procedure

Important: Set up all reactions on ice.

Note: Prepare a separate reaction mix for each locus (HLA-A, HLA-B and HLA-C).

1. Thaw 10x LongRange PCR Buffer, dNTP mix, Nuclease Free H2O, and primer solutions. Mix the solutions thoroughly and centrifuge briefly before use.

2. Prepare a reaction mix as shown in Table 2.

The reaction mix typically contains all of the components needed for PCR except the template DNA. Prepare a volume of reaction mix at least 10% greater than that required for the total number of PCR assays to be performed.

The optimal amount of template DNA to use in the reaction is 100 ng. However, template DNA in the range of 50–200 ng (in 1–5 µl) can be used without affecting results. To streamline the process, validate your DNA purification procedure so that you can use a set volume corresponding to 50–200 ng DNA.


Table 2. Composition of reaction mix for amplification of HLA-A, HLA-B, and HLA-C loci amplification (prepare a separate reaction mix for each locus)

Component Volume in each reaction Final concentration

LongRange PCR Buffer with Mg2+, 10x

2.5 µl 1x; 2.5 mM Mg2+

dNTP mix (10 mM each) 1.25 µl 500 µM of each dNTP

HLA Class I

Amplification Primers

(red cap)

1 µl –

LongRange PCR Enzyme Mix 0.4 µl 2 units per reaction

Nuclease Free H2O Variable (14.85–18.85 µl)

–

Template DNA

Added at step 5

Variable (1–5 µl)

50–200 ng per reaction (optimal 100 ng)

Total volume 25 µl

3. Mix the reaction mix thoroughly, and centrifuge briefly.

4. Dispense the reaction mix into each PCR tube.

The appropriate volume is 25 µl minus the amount of DNA added in the next step.

5. Add 1–5 µl template DNA (50–200 ng) to each tube containing reaction mix.

The final volume must be 25 µl.

The optimal amount of template DNA to use in the reaction is 100 ng. However, template DNA in the range of 50–200 ng (in 1–5 µl) can be used without affecting results.

6. Program the thermal cycler according to the manufacturer’s instructions, using the conditions outlined in Table 3.

Table 3. Cycling protocol for HLA Class I amplification

Comments

Initial activation step:

3 min 95°C Initial denaturation of template DNA.

3-step cycling:

Denaturation 15 s 95°C Do not exceed this temperature.

Annealing 30 s 65°C

Extension 3 min 68°C PCR product sizes

3.1 kb to 3.4 kb

Number of cycles 35

Final extension: 10 min 68°C

End of PCR cycling: Indefinite 4°C

7. Important: For a simplified hot start, place the tubes immediately into a thermal cycler that is heated to 95°C and start the cycling program as outlined in Table 3.

Use the simplified hot start to ensure PCR specificity.

After amplification, samples can be stored overnight at 2–8°C. Clean-up of the PCR products (page 33) should be carried out within 24 h.

8. Confirm the PCR products using an appropriate detection system such as agarose gel electrophoresis. Prepare a 1% w/v agarose gel according to your laboratory protocol, and analyze 3 µl of each PCR assay.

See Table 3 for approximate size of PCR products.

9. Proceed with “Protocol 2: ”, page 30.


Protocol 1B HLA-DRB Loci Amplification

This protocol is for amplification of part of the HLAHLAHLAHLA----DRBDRBDRBDRB1111, HLA, HLA, HLA, HLA----DRB3, DRB3, DRB3, DRB3, HLAHLAHLAHLA----DRB4, and/or HLADRB4, and/or HLADRB4, and/or HLADRB4, and/or HLA----DRB5DRB5DRB5DRB5 genes using the QIAGEN LongRange PCR Kit and the SBTexcellerator® HLA-DRB1 Kit and/or the SBTexcellerator® HLA-DRB3/4/5 Kit.

Procedure


Note: Prepare a separate reaction mix for each locus (HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5).





Table 4. Composition of reaction mix for HLA-DRB loci amplification (prepare a separate reaction mix for each locus)



2.5 µl 1x; 2.5 mM Mg2+


HLA-DRB1, HLA-DRB3, HLA-DRB4, or HLA-DRB5 Amplification Primers

(red cap)

1 µl –



–

Template DNA

Added at step 14



Total volume 25 µl









Table 5. Cycling protocol for HLA-DRB loci amplification

Comments



3-step cycling:



Extension 5 min 68°C PCR product sizes:

3.7 to 4.8 kb for HLA-DRB1

3.8 kb for HLA-DRB3

0.4 kb exon 2 & 1.3 kb exon 3 for HLA-DRB4

4.0 kb for HLA-DRB5

(see figure 1, page 20)

Number of cycles 35







See Table 5 for approximate sizes of PCR products. See Figure 1 below for an example of agarose-gel analysis of HLA-DRB3, HLA-DRB4, and HLA-DRB5 loci.

Figure 1. Locus-specific amplification for HLA-DRB3, HLA-DRB4, and HLA-DRB5. PCR products were analyzed on an agarose gel. M: GelPilot® 200 bp Ladder.



Protocol 1C: HLA-DQA1 Locus Amplification

This protocol is for amplification of part of the HLAHLAHLAHLA----DQDQDQDQAAAA1111 gene using the QIAGEN LongRange PCR Kit and the SBTexcellerator® HLA-DQA1 Kit.

Amplification primers sets QA1*01/3 and QA1*02/4/5/6 are provided for typing allele groups HLA-DQA1*01/03 and HLA-DQA*02/04/05/06, respectively. Both amplification reactions should be performed.

Procedure


Note: Prepare a separate reaction mix for QA1*01/3 and QA1*02/4/5/6 amplification.

19. Thaw 10x LongRange PCR Buffer, dNTP mix, Nuclease Free H2O, and both QA1*01/3 and QA1*02/4/5/6 primer solutions. Mix the solutions thoroughly and centrifuge briefly before use.

20. Prepare a reaction mix as shown in Table 6. Prepare a solution for both QA1*01/3 and QA1*02/4/5/6!



Table 6. Composition of reaction mix for HLA-DQA1 locus amplification (prepare a separate reaction mix for QA1*01/3 and QA1*02/4/5/6 amplification)



2.5 µl 1x; 2.5 mM Mg2+


QA1*01/3

QA1*02/4/5/6

Amplification Primers

(red cap)

1 µl



–

Template DNA

Added at step 23



Total volume 25 µl









Table 7. Cycling protocol for HLA-DQA1 locus amplification

Comments



3-step cycling:




5.4 kb to 5.8 kb

Number of cycles 35









Protocol 1D: HLA-DQB1 Locus Amplification

This protocol is for amplification of part of the HLAHLAHLAHLA----DQB1DQB1DQB1DQB1 gene using the QIAGEN LongRange PCR Kit and the SBTexcellerator® HLA-DQB1 Kit.

Procedure


Note:Note:Note:Note: addition of Q-Solution is required!


29. Prepare a reaction mix as shown in Table 6. Please note that the addition of Q-Solution is required!




Table 6. Composition of reaction mix for HLA-DQB1 locus amplification



2.5 µl 1x; 2.5 mM Mg2+


HLA-DQB1 Amplification Primers (red cap)

1 µl –


QQQQ----Solution, 5xSolution, 5xSolution, 5xSolution, 5x 5 µl5 µl5 µl5 µl 1x1x1x1x


–

Template DNA

Added at step 32



Total volume 25 µl








Table 7. Cycling protocol for HLA-DQB1 locus amplification

Comments



3-step cycling:




3.7 kb to 4.1 kb

Number of cycles 35










Protocol 1E: HLA-DPB1 Locus Amplification

This protocol is for amplification of part of the HLAHLAHLAHLA----DPB1DPB1DPB1DPB1 gene using the QIAGEN LongRange PCR Kit and the SBTexcellerator® HLA-DPB1 Kit.

Procedure






Table 8. Composition of reaction mix for HLA-DPB1 locus amplification



2.5 µl 1x; 2.5 mM Mg2+


HLA-DPB1 Amplification Primers (red cap)

1 µl –



–

Template DNA

Added at step 41



Total volume 25 µl

39. Mix the reaction mixture thoroughly, and centrifuge briefly.







Table 9. Cycling protocol for HLA-DPB1 locus amplification

Comments



3-step cycling:




2.9 kb exon 1 & 5.7 kb exons 2–5

Number of cycles 35






Protocol 2: PCR product Clean-up

The PCR products are treated with Exonuclease I and Shrimp Alkaline Phosphatase (ExoI/SAP) prior to sequencing to degrade unincorporated primers and to dephosphorylize residual nucleotides. As an alternative to PCR product clean-up using the ExoI/SAP, the QIAquick PCR Purification Kit can also be used.

Important note before starting

� Set up the reactions on ice.

Procedure

1. Prepare an ExoI/SAP master mix in a sterile microfuge tube according to table 10.

Table 10. Composition of reaction mix for amplicon clean-up

N=1 N=10 N=25

ExoI I 0.5 µl 5 µl 12.5 µl

SAP 1 µl 10 µl 25 µl

Total 1.5 µl 15 µl 37.5 µl

2. Add 1.5 µl ExoI/SAP Master Mix to each of the PCR products.

3. Gently vortex the mixture to produce a homogeneous reaction and spin down briefly.


Table 11. Cycling protocol for amplicon ExoI/SAP clean-up

Time Temperature

Activation step: 30 min 37°C

Inactivation step: 20 min 80°C

Cooling: Indefinite 4°C

5. Proceed with “Protocol 3: Sequencing of HLA Loci”, page 31


Protocol 3: Sequencing of HLA Loci

This protocol is for sequencing of both HLA Class I and HLA Class II loci. This procedure is optimized for BigDye Terminator chemistry, with subsequent analysis on ABI PRISM 3100 or Applied Biosystems 3130 Genetic Analyzers, or ABI PRISM 3700 or Applied Biosystems 3730 DNA Analyzers.


� Set up the reactions on ice.

� Briefly centrifuge the tubes containing the sequencing primers (yellow and green caps) before opening.

� Read the BigDye Terminator protocol manual, paying particular attention to the “Safety Information” and “Important Notes” sections before beginning the procedure.

Procedure

1. Prepare a sequencing master mix according to Table 12.

The sequencing master mix should contain all of the components needed for all sequence tests to be performed. Prepare a volume of sequencing master mix at least 10% greater than that required for the total number of sequencing assays to be performed.

Note: Prepare a separate sequencing mix for each sequencing primer.

Table 12. Composition of sequencing master mix for sequencing of HLA loci (prepare a separate master mix for each sequencing primer)

Component Volume in each reaction

BDT Ready Reaction Premix, 2.5x 1 µl

BDT Buffer, 5x 1.5 µl

Nuclease Free H2O 5.5 µl

Sequencing primer (yellow or green cap) 1 µl

Total 9 µl

2. Gently mix the sequencing master mix and centrifuge briefly.

3. Dispense 9 µl of the sequencing master mix into each PCR tube.

4. Add 1 µl purified PCR product to each tube containing sequencing master mix.

The final volume is 10 µl.


Table 13. Cycling protocol for sequencing HLA loci

Comments

Initial denaturation: 10 s 96°C Initial denaturation of template DNA.

3-step cycling:

Denaturation 10 s 96°C


Extension 2 min 60°C

Number of cycles 25


6. Place the tubes immediately into a thermal cycler and start the cycling program as outlined in Table 13.

7. After the sequencing reaction is finished, proceed with “Protocol 4: Clean-up and Analysis of HLA Sequencing Products”, page 33.


Protocol 4: Clean-up and Analysis of HLA Sequencing Products

This procedure uses the Sephadex size-exclusion method for clean-up of HLA sequencing products. Sequencing products are purified over a Sephadex column to remove unincorporated sequence primers and free nucleotides.


� Prepare Sephadex at least 3 hours before use.

� As an alternative to clean-up using sephadex, other equivalent size-exclusion methods (e.g. Qiagen DyeEx Kits) or ethanol precipitation of sequencing products can be used.

Preparation Procedure (3 hours before use)

1. Fill the wells of the 45 µl Column Loader with Sephadex G-50.

Remove any excess powder by scraping the remaining Sephadex G-50 with the Multiscreen Column Loader Scraper.

2. Lay a Multiscreen-HV filter plate upside-down on top of the filled 45 µl Column Loader and turn it upside-down so that the filter plate is filled with Sephadex G-50.

3. Add 300 µl deionised water to each well with a multichannel pipette. Be sure to avoid air bubbles during pipetting.

4. Steep the Sephadex G-50 for at least 3 hours at room temperature covered by the lid.

Ensure the sephadex does not dry out due to excessive heat and/or airflow. Steeped Sephadex can be stored for a week at 4°C. Clean the Column Loader by tapping the plate up side down and remove residual powder with a dry tissue.

Clean-up of sequencing products:

5. Put the Multiscreen-HV filter plate, containing the steeped Sephadex G-50, on a 96-well round bottom micro plate placing the Multiscreen Centrifuge Align Frame in between.

6. Spin down at 750g for 5 minutes.

7. Discard the water in the 96-well round bottom micro plate and store the plate for future use.

8. Replace the 96-well round bottom micro plate and the Multiscreen Centrifuge Align Frame by the EU Frosted Sub skirted Thin-wall 96 x 0.2 ml plate.

9. Add 10 µl deionised water to each sequencing reaction product using a multichannel pipette.

10. Pipette the sequence sample (20 µl) on the Multiscreen-HV filter plate in the middle of the well.

11. Spin down at 750g for 5 minutes.

12. Load the EU Frosted Sub skirted Thin-wall 96 x 0.2 ml plate containing the samples into the thermal cycler, heat the samples for 2 min at 95°C and subsequently put them on ice until loading in the genetic analyzer.

13. Load the EU Frosted Sub skirted Thin-wall 96 x 0.2 ml plate containing the samples into the genetic analyzer and run it according to the manufacturer’s instructions.

Note: Matrix standards must be run for first-time use of BigDye Terminator reactions. Follow the instructions provided in the BigDye Terminator Cycle Sequencing Kit or by the manufacturer.

SBTexcellerator® HLA Kits have been tested with various polymers and sequencing chemistries.

If sequencing is not possible the same day; seal the plates with an adhesive seal and store the plate at -20°C.

14. Process the collected raw sequence data with the sequencing analysis software SBTengine® or other suitable HLA typing software.


Troubleshooting Guide

This troubleshooting guide may be helpful in solving any problems that may arise. For more information, see also the Frequently Asked Questions page at the QIAGEN Technical Support Center: www.qiagen.com/FAQ/FAQList.aspx. The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocols in this Instructions for Use or sample and assay technologies (for contact information visit www.qiagen.com).

Comments and suggestions

Little or no PCR product

a) LongRange PCR Enzyme Mix was not added to the amplification mix or not mixed properly when added

Repeat amplification paying attention to the addition and mixing of LongRange PCR Enzyme Mix with the amplification mix.

b) Cycling conditions not optimal

When using a fast thermal cycler, reduce the ramp rate to 1°C/s.

c) DNA concentration not optimal

Requantify the DNA and adjust to 50 ng/µl. If the sample concentration is below the recommended range and little or no amplification product is visible, sequence the sample. Acceptable sequence and typing may be achievable.

d) Poor-quality or degraded genomic DNA

Run genomic DNA on a 1% agarose gel to evaluate quality.

Purified DNA should have an A260/A280 ratio between 1.7 and 1.9.

e) Weak amplification of HLA-DRB4 exon 3

Use the recommended amount of purified PCR product for sequencing, even if amplification of exon 3 is weak. Use 63°C annealing instead of 65°C as described in Table 5, page 19. Acceptable sequencing data should still be obtained.


Unusual PCR products

a) Two PCR products visible after amplification of HLA-DRB1, HLA-DRB4, or HLA-DQB1 locus

In a heterozygous sample, 2 bands may appear for the HLA-DRB1 or HLA-DQB1 PCR products due to length polymorphism in intron regions of the HLA-DRB1 or HLA-DQB1 gene.

Amplification of HLA-DRB4 normally results in 2 PCR products (see Figure 1, page 20), except for DRB4*0301N, which does not possess exon 2.

b) HLA-DRB1 or HLA-DQB1 amplicons of different samples have different sizes

Due to length polymorphisms in intron regions of the HLA-DRB1 or HLA-DQB1 gene, amplicons may vary in size in different samples.

Excessive background noise

a) PCR products not cleaned up prior to sequencing

Clean-up the PCR products using the ExoI/SAP clean-up method before using them in the sequencing reaction.

b) No or poor clean-up of sequencing reactions

Be sure to perform clean-up of sequencing reactions using Protocol 4: Clean-up and Analysis of HLA Sequencing Products. Pipette the sample directly onto the center of the gel-bed surface. Do not allow the reaction mixture or the pipette tip to contact the sides of the gel-bed or the sides of the wells of the plates.

c) Signal strength too high See “Excessive signal strength” below.

d) Poor or incorrect matrix Repeat the spectral calibration and re-inject samples.

e) Poor injection Re-inject samples.

f) Injection time set too high Reduce injection time and re-inject. Signal strengths of 100–1500 are optimal. Samples of poor quality may have lower signal strengths but may still be analyzed and typed. Some samples may have signals that are over 1500 and will not have excess background.

g) Peaks shifted or on top of each other

Incorrect mobility file chosen. Choose correct mobility file.



h) Poor sequence quality in one of the HLA-DRB1 exon 2 forward sequences

There are 2 forward sequencing primers for exon 2. Both should be used routinely. Primer R3 is specific for sequencing alleles in group DR1/2/3/5/6/8/10. Primer R4 is specific for sequencing alleles present in group DR4/7/9. With homozygous samples, sequences will be obtained from only one of the primers. In heterozygous samples possessing 2 alleles from the same set of groups (e.g., group DR1 and group DR6), heterozygous sequence data is to be expected from the primer for the relevant groups, and the other primer will generate bad or no sequence. The SBTengine® software will reject this sequence data in most cases, so it does not interfere with typing analysis.

Weak signal

a) Injection time needs to be increased

Repeat sequencing reactions and increase injection time.

b) PCR product concentration too low

Increase the amount of PCR product in the sequencing reaction, and reduce the amount of Nuclease Free H2O proportionately.

Excessive dye blobs

a) No or poor clean-up of sequencing reactions

Be sure to perform clean-up of sequencing reactions using protocol Protocol 4: Clean-up and Analysis of HLA Sequencing Products. Pipette the sample directly onto the center of the gel-bed surface. Do not allow the reaction mixture or the pipette tip to contact the sides of the gel-bed or the sides of the wells of the plates.

b) Poor sequencing reaction due to error in pipetting or weak amplification product

Be sure that both the cleaned-up amplicon and the correct sequencing mix are added and combined. In the case of weak amplification, confirm the intensity of the amplicon by running an agarose gel.


Excessive signal strength

a) PCR product too concentrated

Dilute the PCR product with Nuclease Free H2O before sequencing.

b) Too much BDT Ready Reaction Premix in the sequencing reaction

Reduce the amount of BDT Reaction Premix and adjust the amount of BDT Buffer according to the manufacturer’s instructions.

c) Injection time set too high Reduce injection time and reinject. Signal strengths of 100–1500 are optimal. Samples of poor quality may have lower signal strengths but may still be analyzed and typed. Some samples may have signals that are over 1500 and will not have excess background.


Appendix A: Control of Contamination

It is extremely important to include at least one negative control in every PCR setup that lacks template nucleic acid to detect possible contamination.

General physical precautions

� Separate the working areas for setting up the PCR amplification mix and DNA handling, including the addition of starting template, PCR product analysis, or plasmid preparation. Ideally, use separate rooms.

� Use a separate set of pipettes for the PCR amplification mix. Use of pipette tips with hydrophobic filters is strongly recommended.

� Prepare and freeze small aliquots of primer solutions and dNTP mix. Use of fresh Nuclease Free H2O is strongly recommended.

� In case of contamination, laboratory benches, apparatus, and pipettes can be decontaminated by cleaning them with a 1/10 dilution of a commercial bleach solution.*† Afterwards, the benches and pipettes must be rinsed with Nuclease Free H2O.

General chemical precautions

� PCR stock solutions can also be decontaminated using UV light. This method is laborious, however, and its efficiency is difficult to control and cannot be guaranteed. We recommend storing solutions in small aliquots and using fresh aliquots for each PCR.

� Another approach to prevent amplification of contaminating DNA is to treat individual reaction mixtures with DNAse I† or restriction enzymes† that cut between the binding sites of the amplification primers used, before adding the template DNA sample.

* Most commercial bleach solutions are approximately 5.25% sodium hypochlorite. Sodium hypochlorite is an irritant and should be handled with caution.

† When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate material safety data sheets (MSDSs), available from the product supplier.

Ordering Information

QIAGEN’s sequencing Based typing (SBT) products are supported either directly by QIAGEN subsidiaries, by your local QIAGEN distributor or reseller. Please contact your local HLA distributor or QIAGEN Customer Care Service at +49 2103-29-12250.

Limited License Agreement

Use of this product signifies the agreement of any purchaser or user of the SBTexcellerator® HLA Kits to the following terms:

1. The SBTexcellerator® HLA Kits may be used solely in accordance with the GenDx SBTexcellerator® Instructions For Use and for use with components contained in the Kit only. Genome Diagnostics B.V. grants no license under any of its intellectual property to use or incorporate the enclosed components of this Kit with any components not included within this Kit except as described in the GenDx SBTexcellerator® Instructions For Use and additional protocols available at www.GenDx .com.

2. Other than expressly stated licenses, Genome Diagnostics B.V. makes no warranty that this Kit and/or its use(s) do not infringe the rights of third-parties.

3. This Kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.

4. Genome Diagnostics B.V. specifically disclaims any other licenses, expressed or implied other than those expressly stated.

5. The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. Genome Diagnostics B.V. may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Kit and/or its components.

For updated license terms, see www.GenDx.com.

© 2011 Genome Diagnostics B.V., all rights reserved.

Trademarks: QIAGEN®, QIAquick®, DyeEx® (QIAGEN Group); ABI PRISM®, BigDye® (Applera Corporation); SBTengine®®, SBTexcellerator®® (Genome Diagnostics).

SBTexcellerator® HLA Kits are licensed from Genome Diagnostics BV. QIAGEN acts as a co-exclusive distributor of SBTengine® software.


Notes

3840000 07/2011 1068327 Sample & Assay Technologies

DE Die Gebrauchsanweisungen und Protokollblätter können in deutscher Sprache von der folgenden Website heruntergeladen werden: www.gendx.com/handbooks. Wenn Sie diese Website nicht erreichen können, senden wir Ihnen gerne die einschlägige Übersetzung per E-Mail, Fax oder Post zu. Kontaktieren Sie bitte Genome Diagnostics B.V. unter der Telefonnummer +31 302 523 799.

EN The english instructions for use and protocol sheets can be downloaded at the following web site: www.gendx.com/handbooks. If you do not have access to this web site, we will gladly send you the relevant translation by e-mail, fax, or post. Please contact Genome Diagnostics B.V. at the telephone number +31 302 523 799.

ES Puede descargarse las instrucciones de uso y las hojas de protocolo en español en la siguiente página web: www.gendx.com/handbooks. Si no tiene acceso a este sitio web, será un placer enviarle la traducción pertinente por correo electrónico, fax, o correo postal. Póngase en contacto con Genome Diagnostics B.V. en el número de teléfono +31 302 523 799.

FR Les guides d'utilisation en français ainsi que les fiches de protocole, peuvent être téléchargés sur le site Web suivant : www.gendx.com/handbooks. Si vous n'avez pas accès à ce site web, nous vous enverrons volontiers la traduction appropriée par e-mail, fax ou courrier. Veuillez contacter Genome Diagnostics BV au numéro de téléphone +31 302 523 799.

IT Le istruzioni di utilizzo e i protocolli in italiano possono essere scaricati dal sito Web: www.gendx.com/handbooks. Se non è possibile accedere al sito, potremo inviare la relativa traduzione per e-mail, fax o posta. Contattare Genome Diagnostics BV al numero di telefono +31 302 523 799.

instructions for use gendx sbt excellerator® · eighth edition july 2011 sample & assay...

Documents