Eighth Edition
July 2011
Instructions For Use
GenDx SBTexcellerator®
For high-resolution HLA Sequencing Based Typing (SBT)
For Research Use Only
Genome Diagnostics B.V.
Phone:
+31 302 523 799
Email:
[email protected]
Web:
www.gendx.com
Address:
Yalelaan 48
3584 CM Utrecht
Distributed by
the Netherlands
Sample & Assay Technologies
For Research Use Only
Not for use in diagnostic procedures. No claim or representation is intended to
provide information for the diagnosis, prevention or treatment of a disease.
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].
Page 2
Eighth Edition
July 2011
Contents
For Research Use Only
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
Safety Information
9
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
Page 3
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
Legal Manufacturer
Add liquid
Page 4
Eighth Edition
July 2011
Kit Content
For HLA Class I
SBTexcellerator® HLA-A Kits
Catalog no.
Core Kit (50) Extd. Kit (50)
4100034
4100134
50
50
HLA-A Amplification Primers (red cap)
1 tube
–
HLA-A Sequencing Primers (yellow cap)
Multiple tubes*
–
–
Multiple tubes*
1 tube
1 tube
HLA-A Protocol Sheet
3000101
–
Instructions For Use
3340000
–
Number of reactions
HLA-A GSSP Primers (green cap)
Nuclease Free H2O (clear cap)
SBTexcellerator® HLA-B Kits
Catalog no.
Number of reactions
HLA-B Amplification Primers (red cap)
HLA-B Sequencing Primers (yellow cap)
HLA-B GSSP Primers (green cap)
Core Kit (50) Extd. Kit (50)
4101034
4101134
50
50
1 tube
–
Multiple tubes* Multiple tubes*
–
Multiple tubes*
1 tube
1 tube
HLA-B Protocol Sheet
3000201
–
Instructions for Use
3340000
–
Nuclease Free H2O (clear cap)
* See the protocol sheet for primer details.
Page 5
SBTexcellerator® HLA-C Kits
Catalog no.
Number of reactions
HLA-C Amplification Primers (red cap)
HLA-C Sequencing Primers (yellow cap)
HLA-C GSSP Primers (green cap)
Core Kit (50) Extd. Kit (50)
4102034
4102134
50
50
1 tube
–
Multiple tubes* Multiple tubes*
–
Multiple tubes*
1 tube
1 tube
HLA-C Protocol Sheet
3000301
–
Instructions for Use
3340000
–
Nuclease Free H2O(clear cap)
*
See the protocol sheet for primer details.
Page 6
Eighth Edition
July 2011
For HLA Class II
SBTexcellerator® HLADRB Kits
HLA-DRB1
Core (50)
HLA-DRB1
Extd. (50)
HLA-DRB3/4/5
(50)
4105034
4105134
4105334
50
50
50
HLA-DRB1 Amplification
Primers (red cap)
1 tube
–
–
HLA-DRB3 Amplification
Primers (red cap)
–
–
1 tube
HLA-DRB4 Amplification
Primers (red cap)
–
–
1 tube
HLA-DRB5 Amplification
Primers (red cap)
–
–
1 tube
Multiple
tubes*
1 tube
–
–
–
Multiple tubes*
1 tube
Multiple
tubes*
–
–
–
1 tube
1 tube
1 tube
1 tube
HLA-DRB Protocol Sheet
3000601
–
3000701
Instructions for Use
3340000
–
3340000
Catalog no.
Number of reactions
HLA-DRB1 Sequencing
Primers (yellow cap)
HLA-DRB3/4/5
Sequencing Primers
(yellow cap)
HLA-DRB1 GSSP primers
(green cap)
HLA-DRB3/4/5 GSSP
primers (green cap)
Nuclease Free H2O
(clear cap)
* See the protocol sheet for primer details.
Page 7
SBTexcellerator® HLA-DQA1 Kits
Catalog no.
Number of reactions
HLA-DQA1 Amplification Primers (red cap)
Core Kit (50)
Extd. Kit (50)
4106034
4106134
50
50
2 tubes
–
HLA-DQA1 Sequencing Primers (yellow cap) Multiple tubes* Multiple tubes*
Nuclease Free H2O (clear cap)
1 tube
1 tube
HLA-DQA1 Protocol Sheet
3000801
–
Instructions for Use
3340000
–
Core Kit (50)
Extd. Kit (50)
4104034
4104134
50
50
1 tube
–
SBTexcellerator® HLA-DQB1 Kits
Catalog no.
Number of reactions
HLA-DQB1 Amplification Primers (red cap)
HLA-DQB1 Sequencing Primers (yellow cap) Multiple tubes*
HLA-DQB1 GSSP Primers (green cap)
–
–
Multiple tubes*
1 tube
1 tube
HLA-DQB1 Protocol Sheet
3000501
–
Instructions for Use
3340000
–
Nuclease Free H2O (clear cap)
* See the protocol sheet for primer details.
Page 8
Eighth Edition
SBTexcellerator® HLA-DPB1 Kits
Catalog no.
Number of reactions
HLA-DPB1 Amplification Primers (red cap)
HLA-DPB1 Sequencing Primers (yellow cap)
HLA-DPB1 GSSP Primers (green cap)
July 2011
Core Kit (50) Extd. Kit (50)
4103034
4103134
50
50
1 tube
–
Multiple tubes* Multiple tubes*
–
Multiple tubes*
1 tube
1 tube
HLA-DPB1 Protocol Sheet
3000401
–
Instructions for Use
3340000
–
Nuclease Free H2O (clear cap)
* See the protocol sheet for primer details.
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
For Research Use Only. Not for use in diagnostic procedures. No claim or
representation is intended to provide information for the diagnosis, prevention,
or treatment of a disease.
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.
Page 9
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). The specimen material is human genomic DNA.
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 highresolution 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.
Page 10
Eighth Edition
July 2011
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
Briefly centrifuge the tubes containing the amplification primers (red caps)
and sequencing primers (yellow and green caps) before opening for the
first time. Resuspend each primer in Nuclease Free H2O (provided), using
the resuspension
resuspension volumes listed in Table 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
Page 11
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.
Page 12
Eighth Edition
July 2011
For clean-up and analysis of HLA sequencing products
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.
Page 13
Protocol 1A: HLA Class I Loci Amplification
This protocol is for amplification of HLAHLA-A, HLAHLA-B, or HLAHLA-C 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.
Page 14
Eighth Edition
July 2011
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)
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
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
Component
Amplification Primers
(red cap)
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.
Page 15
Table 3. Cycling protocol for HLA Class I amplification
Comments
Initial activation
step:
3 min
95°C
Initial denaturation of
template DNA.
Denaturation
15 s
95°C
Do not exceed this
temperature.
Annealing
30 s
65°C
Extension
3 min
68°C
3-step cycling:
PCR product sizes
3.1 kb to 3.4 kb
Number of cycles
35
Final extension:
10 min
End of PCR cycling: Indefinite
68°C
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.
Page 16
Eighth Edition
July 2011
Protocol 1B HLA-DRB Loci Amplification
This protocol is for amplification of part of the HLAHLA-DRB1,
DRB1, HLAHLA-DRB3,
HLAHLA-DRB4, and/or HLAHLA-DRB5 genes using the QIAGEN LongRange PCR Kit
and the SBTexcellerator® HLA-DRB1 Kit and/or the SBTexcellerator®
HLA-DRB3/4/5 Kit.
Procedure
Important: Set up all reactions on ice.
Note: Prepare a separate reaction mix for each locus (HLA-DRB1,
HLA-DRB3, HLA-DRB4, and HLA-DRB5).
10. Thaw 10x LongRange PCR Buffer, dNTP mix, Nuclease Free H2O, and
primer solutions. Mix the solutions thoroughly and centrifuge briefly
before use.
11. Prepare a reaction mix as shown in Table 4.
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.
Page 17
Table 4. Composition of reaction mix for HLA-DRB loci amplification
(prepare a separate reaction mix for each locus)
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-DRB1, HLA-DRB3,
HLA-DRB4, or HLA-DRB5
Amplification Primers
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 14
Variable
(1–5 µl)
50–200 ng per
reaction (optimal
100 ng)
Total volume
25 µl
Component
(red cap)
12. Mix the reaction mix thoroughly, and centrifuge briefly.
13. Dispense the reaction mix into each PCR tube.
The appropriate volume is 25 µl minus the amount of DNA added in the
next step.
14. 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.
15. Program the thermal cycler according to the manufacturer’s
instructions, using the conditions outlined in Table 5.
Page 18
Eighth Edition
July 2011
Table 5. Cycling protocol for HLA-DRB loci amplification
Comments
Initial activation
step:
3 min
95°C
Initial denaturation of
template DNA.
Denaturation
15 s
95°C
Do not exceed this
temperature.
Annealing
30 s
65°C
Extension
5 min
68°C
3-step cycling:
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
Final extension:
10 min
End of PCR cycling: Indefinite
68°C
4°C
16. 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 5.
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 30) should be carried out within 24 h.
17. 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 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.
Page 19
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.
18. Proceed with “Protocol 2: ”, page 30.
Page 20
Eighth Edition
July 2011
Protocol 1C: HLA-DQA1 Locus Amplification
This protocol is for amplification of part of the HLAHLA-DQA
DQA1 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
Important: Set up all reactions on ice.
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!
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.
Page 21
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)
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
QA1*01/3
1 µl
Component
QA1*02/4/5/6
Amplification Primers
(red cap)
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 23
Variable
(1–5 µl)
50–200 ng per
reaction (optimal
100 ng)
Total volume
25 µl
21. Mix the reaction mix thoroughly, and centrifuge briefly.
22. Dispense the reaction mix into each PCR tube.
The appropriate volume is 25 µl minus the amount of DNA added in the
next step.
23. 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.
24. Program the thermal cycler according to the manufacturer’s
instructions, using the conditions outlined in Table 7.
Page 22
Eighth Edition
July 2011
Table 7. Cycling protocol for HLA-DQA1 locus amplification
Comments
Initial activation
step:
3 min
95°C
Initial denaturation of
template DNA.
Denaturation
15 s
95°C
Do not exceed this
temperature.
Annealing
30 s
65°C
Extension
6 min
68°C
3-step cycling:
PCR product sizes
5.4 kb to 5.8 kb
Number of cycles
35
Final extension:
10 min
End of PCR cycling: Indefinite
68°C
4°C
25. 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 7.
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 30) should be carried out within 24 h.
26. 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 7 for approximate size of PCR products.
27. Proceed with “Protocol 2: ”, page 30.
Page 23
Protocol 1D: HLA-DQB1 Locus Amplification
This protocol is for amplification of part of the HLAHLA-DQB1 gene using the
QIAGEN LongRange PCR Kit and the SBTexcellerator® HLA-DQB1 Kit.
Procedure
Important: Set up all reactions on ice.
Note: addition of Q-Solution is required!
28. Thaw 10x LongRange PCR Buffer, dNTP mix, Nuclease Free H2O, and
primer solutions. Mix the solutions thoroughly and centrifuge briefly
before use.
29. Prepare a reaction mix as shown in Table 6. Please note that the
addition of Q-Solution is required!
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.
Page 24
Eighth Edition
July 2011
Table 6. Composition of reaction mix for HLA-DQB1 locus amplification
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-DQB1 Amplification
Primers (red cap)
1 µl
–
LongRange PCR Enzyme Mix
0.4 µl
2 units per reaction
Q-Solution, 5x
5 µl
1x
Nuclease Free H2O
Variable
(9.85–13.85 µl)
–
Template DNA
Added at step 32
Variable
(1–5 µl)
50–200 ng per
reaction (optimal
100 ng)
Total volume
25 µl
Component
30. Mix the reaction mix thoroughly, and centrifuge briefly.
31. Dispense the reaction mix into each PCR tube.
The appropriate volume is 25 µl minus the amount of DNA added in the
next step.
32. 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.
33. Program the thermal cycler according to the manufacturer’s
instructions, using the conditions outlined in Table 7.
Page 25
Table 7. Cycling protocol for HLA-DQB1 locus amplification
Comments
Initial activation
step:
3 min
95°C
Initial denaturation of
template DNA.
Denaturation
15 s
95°C
Do not exceed this
temperature.
Annealing
30 s
65°C
Extension
4 min
68°C
3-step cycling:
PCR product sizes
3.7 kb to 4.1 kb
Number of cycles
35
Final extension:
10 min
End of PCR cycling: Indefinite
68°C
4°C
34. 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 7.
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 30) should be carried out within 24 h.
35. 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 7 for approximate size of PCR products.
36. Proceed with “Protocol 2: ”, page 30.
Page 26
Eighth Edition
July 2011
Protocol 1E: HLA-DPB1 Locus Amplification
This protocol is for amplification of part of the HLAHLA-DPB1 gene using the
QIAGEN LongRange PCR Kit and the SBTexcellerator® HLA-DPB1 Kit.
Procedure
Important: Set up all reactions on ice.
37. Thaw 10x LongRange PCR Buffer, dNTP mix, Nuclease Free H2O, and
primer solutions. Mix the solutions thoroughly and centrifuge briefly
before use.
38. Prepare a reaction mix as shown in Table 8.
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 8. Composition of reaction mix for HLA-DPB1 locus amplification
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-DPB1 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 41
Variable
(1–5 µl)
50–200 ng per
reaction (optimal
100 ng)
Total volume
25 µl
Component
Page 27
39. Mix the reaction mixture thoroughly, and centrifuge briefly.
40. Dispense the reaction mix into each PCR tube.
The appropriate volume is 25 µl minus the amount of DNA added in the
next step.
41. 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.
42. Program the thermal cycler according to the manufacturer’s
instructions, using the conditions outlined in Table 9.
Table 9. Cycling protocol for HLA-DPB1 locus amplification
Comments
Initial activation
step:
3 min
95°C
Initial denaturation of
template DNA.
Denaturation
15 s
95°C
Do not exceed this
temperature.
Annealing
30 s
65°C
Extension
6 min
68°C
3-step cycling:
PCR product sizes
2.9 kb exon 1 & 5.7 kb
exons 2–5
Number of cycles
35
Final extension:
10 min
End of PCR cycling: Indefinite
68°C
4°C
43. 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 9.
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 30) should be carried out within 24 h.
Page 28
Eighth Edition
July 2011
44. 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 9 for approximate size of PCR products.
45. Proceed with “Protocol 2: ”, page 30.
Page 29
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
0.5 µl
5 µl
12.5 µl
SAP
1 µl
10 µl
25 µl
1.5 µl
15 µl
37.5 µl
Total
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.
4. Program the thermal cycler according to the manufacturer’s
instructions, using the conditions outlined in Table 11.
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
Page 30
Eighth Edition
July 2011
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.
Important notes before starting
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
BDT Ready Reaction Premix, 2.5x
Volume in each reaction
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.
Page 31
5. Program the thermal cycler according to the manufacturer’s
instructions, using the conditions outlined in Table 13.
Table 13. Cycling protocol for sequencing HLA loci
Comments
Initial denaturation:
10 s
96°C
Denaturation
10 s
96°C
Annealing
10 s
50°C
Extension
2 min
60°C
Number of cycles
25
End of PCR cycling:
Indefinite 4°C
Initial denaturation of template
DNA.
3-step cycling:
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.
Page 32
Eighth Edition
July 2011
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.
Important notes before starting
Prepare Sephadex at least 3 hours before use.
As an alternative to clean-up using sephadex, other equivalent sizeexclusion 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.
Page 33
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.
Page 34
Eighth Edition
July 2011
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.
Page 35
Comments and suggestions
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 gelbed 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.
Page 36
Eighth Edition
July 2011
Comments and suggestions
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
Be sure that both the cleaned-up amplicon
due to error in pipetting or and the correct sequencing mix are added
weak amplification product and combined. In the case of weak
amplification, confirm the intensity of the
amplicon by running an agarose gel.
Page 37
Comments and suggestions
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.
Page 38
Eighth Edition
July 2011
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.
Page 39
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.
Page 40
Eighth Edition
July 2011
Notes
Page 41
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Eighth Edition
July 2011
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Sample & Assay Technologies