Instructions for Use

Instructions for Use
Instructions for Use
PCR Amplification and Sequencing of HLA Class I and II Loci
Version No: 15.0
Issue Date: August 2015
EC
Conexio Genomics Pty Ltd
2/49 Buckingham Dr
Wangara 6065
Western Australia
Australia
REP
Qarad bvba
Cipalstraat 3
B-2440 Geel
Belgium
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For In-Vitro Diagnostic Use
Contents
PRINCIPLE ..............................................................................................................................3
INTENDED USE ......................................................................................................................3
KIT COMPOSITION ..............................................................................................................4
STORAGE REQUIREMENTS ...............................................................................................9
MATERIALS, REAGENTS AND EQUIPMENT NOT SUPPLIED ..................................9
SAMPLE REQUIREMENTS................................................................................................10
WARNINGS AND SAFETY PRECAUTIONS ...................................................................11
SYMBOLS ..............................................................................................................................11
PROCEDURE .........................................................................................................................12
1.
2.
3.
4.
5.
6.
7.
PCR .......................................................................................................................................... 12
AGAROSE GEL ELECTROPHORESIS ............................................................................................ 12
PURIFICATION OF PCR PRODUCT .............................................................................................. 13
SEQUENCING REACTION ........................................................................................................... 14
PURIFICATION OF SEQUENCING REACTION PRODUCTS.............................................................. 15
DENATURATION & ELECTROPHORESIS OF SEQUENCING REACTION PRODUCTS ........................ 16
EDITING AND ANALYSIS OF ELECTROPHEROGRAMS .................................................................. 17
PERFORMANCE CHARACTERISTICS ...........................................................................18
ACCURACY ......................................................................................................................................... 18
DETECTION LIMIT ............................................................................................................................... 19
SPECIFICITY ........................................................................................................................................ 19
LIMITATIONS AND CAUTIONS .......................................................................................19
LICENSE ................................................................................................................................20
BIBLIOGRAPHY...................................................................................................................20
TROUBLESHOOTING .........................................................................................................21
RELATED PRODUCTS ........................................................................................................24
SUPPORT AND CONTACT DETAILS ..............................................................................25
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For In-Vitro Diagnostic Use
Principle
The HLA Sequencing Based Typing (SBT) procedure described here was originally
developed by D. Sayer in 20011 and developed into a single tube assay in 20042. The
procedure involves the initial amplification of the target sequence followed by enzymatic
treatment to remove unincorporated primers and dNTPs. The amplicon is then used as a
template for direct automated fluorescent DNA sequencing using customized sequencing
primers and the Big Dye® Terminator sequencing chemistry available from Applied
Biosystems™ by Life Technologies™. The extension products are purified according to the
ethanol precipitation method and denatured using Hi-Di™ formamide available from Applied
Biosystems™ by Life Technologies™, before separation and detection on an automated
fluorescent DNA sequencer. It is recommended that the resulting data is then analysed with
Assign™ SBT sequence analysis software from Conexio Genomics Pty Ltd3-5.
Intended Use
Conexio Genomics’ SBT Resolver™ HLA SBT kits are used for the typing of HLA Class I
(HLA-A, -B, and -C) and Class II (HLA-DRB1, -DQB1 and -DPB1) genes in a laboratory
setting from genomic DNA. Each kit contains reagents that facilitate the PCR amplification
and DNA sequencing of a given gene. The resultant sequencing data is then interpreted
through the use of Conexio Genomics’ Assign™ SBT software. It should be noted that these
SBT kits are not used for the diagnosis of disease.
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For In-Vitro Diagnostic Use
Kit Composition
Kit
PRE-PCR Contents†
(No of vials)
Catalogue No
POST-PCR Contents
(No of vials)
Class I
HLA-A
XH-PD1.1-2(20)
XH-PD1.1-2(50)
HLA-B
BS-PD2.1-2(20)
20 tests
50 tests
20 tests
DNA POL – HLA-A
1 x 25L
AEX1F
AEX1R
HLA-A MIX
1 x 352L
AEX2F
AEX2R
AEX3F
AEX3R
AEX4F
AEX4R
DNA POL – HLA-A
1 x 60L
AEX1F
AEX1R
HLA-A MIX
1 x 880L
AEX2F
AEX2R
AEX3F
AEX3R
AEX4F
AEX4R
BEX1F
BEX2F
BEX2R
BEX3F
BEX3R
BEX4F
DNA POL – HLA-B
1 x 25L
HLA-B MIX
1 x 352L
1 x 44L each
1 x 110L each
1 x 44L each
BEX4R
BS-PD2.1-2(50)
50 tests
DNA POL – HLA-B
1 x 60L
HLA-B MIX
1 x 880L
BEX1F
BEX2F
BEX2R
BEX3F
BEX3R
BEX4F
1 x 110L each
BEX4R
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For In-Vitro Diagnostic Use
Kit
Catalogue No
HLA-C
HH-PD 3.2-2(20)
PRE-PCR Contents†
(No of vials)
20 tests
POST-PCR Contents
(No of vials)
DNA POL – HLA-C
1 x 25L
CEX1F
CEX1R
HLA-C MIX
1 x 352L
CEX2F
CEX2R
CEX3F
CEX3R
CEX4F
CEX4R
CEX5F
CEX5R
CEX6F
CEX6R
1 x 44L each
CEX7F
HH-PD 3.2-2(50)
50 tests
DNA POL – HLA-C
1 x 60L
CEX1F
CEX1R
HLA-C MIX
1 x 880L
CEX2F
CEX2R
CEX3F
CEX3R
CEX4F
CEX4R
CEX5F
CEX5R
CEX6F
CEX6R
1 x 110L each
CEX7F
Class II
HLA-DRB1
HH-PD5.2-5(20)
20 tests
DNA POL – DRB1
1 x 10L
DRB1EX2F
HLA-DRB1 MIX
1 x 370L
DRB1EX3R-2
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DRB1EX2R-2
RB-TG344-R
1 x 44L
each
For In-Vitro Diagnostic Use
Kit
PRE-PCR Contents†
(No of vials)
Catalogue No
HH-PD5.2-5(50)
LG-PD5.2-7(20)
50 tests
20 tests
POST-PCR Contents
(No of vials)
DNA POL – DRB1
1 x 20L
DRB1EX2F
DRB1EX2R-2
HLA-DRB1 MIX
1 x 920L
DRB1EX3R-2
DNA POL – DRB1
1 x 10L
DRB1EX2F
DRB1EX2R-2
HLA-DRB1 MIX
1 x 370L
DRB1EX3F-7
DRB1EX3R-7
RB-TG344-R
1 x 110L
each
1 x 44L
each
RB-TG344-R
LG-PD5.2-7(50)
50 tests
DNA POL – DRB1
1 x 20L
DRB1EX2F
DRB1EX2R-2
HLA-DRB1 MIX
1 x 920L
DRB1EX3F-7
DRB1EX3R-7
1 x 110L
each
RB-TG344-R
HLA-DQB1
PQ-PD6.2-2(20)
PQ-PD6.2-2(50)
AN-PD6.2-3(20)
20 tests
50 tests
20 tests
DNA POL – DQB1
1 x 10L
DQB1EX2F
DQB1EX2R
HLA-DQB1 MIX
1 x 370L
DQB1EX3F
DQB1EX3R
DNA POL – DQB1
1 x 20L
DQB1EX2F
DQB1EX2R
HLA-DQB1 MIX
1 x 920L
DQB1EX3F
DQB1EX3R
DNA POL – DQB1
1 x 10L
DQB1EX2F
DQB1EX2R-3
HLA-DQB1 MIX
1 x 370L
DQB1EX3F
DQB1EX3R
Page 6 of 25
1 x 44L
each
1 x 110L
each
1 x 44L
each
For In-Vitro Diagnostic Use
Kit
AN-PD6.2-3(50)
HLA-DPB1
PRE-PCR Contents†
(No of vials)
Catalogue No
HH-PD10.1(20)
HH-PD10.1(50)
KD-PD10.2-1(20)
50 tests
20 tests
50 tests
20 tests
POST-PCR Contents
(No of vials)
DNA POL – DQB1
1 x 20L
DQB1EX2F
DQB1EX2R-3
HLA-DQB1 MIX
1 x 920L
DQB1EX3F
DQB1EX3R
DNA POL – DPB1
1 x 10L
DPB1EX2F
DPB1EX2R
HLA-DPB1 MIX
1 x 370L
1 x 44L
each
DNA POL – DPB1
1 x 20L
DPB1EX2F
DPB1EX2R
HLA-DPB1 MIX
1 x 920L
1 x 110L
each
DNA POL – DPB1
1 x 10L
DPB1EX1F
DPB1EX1R
HLA-DPB1 MIX
1 x 370L
DPB1EX2F
DPB1EX2R
DPB1EX3F
DPB1EX3R
DPB1EX4F
DPB1EX4R
DPB1EX5F
DPB1EX5R
1 x 110L
each
1 x 44L
each
PB-AG341-R
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For In-Vitro Diagnostic Use
Kit
PRE-PCR Contents†
(No of vials)
Catalogue No
KD-PD10.2-1(50)
50 tests
POST-PCR Contents
(No of vials)
DNA POL – DPB1
1 x 20L
DPB1EX1F
DPB1EX1R
HLA-DPB1 MIX
1 x 920L
DPB1EX2F
DPB1EX2R
DPB1EX3F
DPB1EX3R
DPB1EX4F
DPB1EX4R
DPB1EX5F
DPB1EX5R
1 x 110L
each
PB-AG341-R
†
The PRE-PCR kit contains a vial of a locus-specific PCR mix (e.g.
primers, along with a single vial of DNA polymerase (e.g.
The POST-PCR kit contains sequencing primers (e.g.
HLA-A MIX
DNA POL – HLA-A
AEX1F
) consisting of PCR buffer, dNTPs, MgCl2, and locus specific PCR
).
).
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For In-Vitro Diagnostic Use
Storage Requirements
The PRE- and POST-PCR boxes may be separated and stored in designated PRE- and POSTPCR freezers. When stored at -20C (temperature range of -15C to -25C is acceptable), the
kit components can be used until the expiry indicated on the outer kit containers and can
tolerate up to 25 freeze-thaw cycles.
Accelerated stability testing for the HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 kits indicated
a shelf life of two and a half years from date of manufacture when stored at -20°C. While
confirmatory real-time testing is underway it is strongly recommended that these kits are
NOT to be used beyond their expiry date.
To maintain optimal kit performance, the kit components should be removed from the -20C
storage location and thawed rapidly at room temperature before use. The kit components, with
the exception of the polymerase, should then be gently vortexed to ensure that the
components of each tube are appropriately mixed after thawing. After use, the
kits/components should be returned immediately to -20C.
Materials, Reagents and Equipment Not Supplied
PCR
1.
Sterile water
2.
Electronic or mechanical pipettes and aerosol-resistant tips
3.
Thermal cycler with heated lid
These kits have been validated using the following thermal cyclers:
MJ Research PTC 225 DNA Engine DYAD™, Applied Biosystems™ by Life
Technologies™ Veriti™ Thermal cycler, Gene Amp® PCR System 9700, and
Eppendorf Mastercycler® Pro.
Use of other thermal cyclers with these kits requires validation by the user.
4.
0.2mL thin-walled thermal cycling reaction tubes (8 well strips or 96 well plates).
Use those recommended for use with your thermal cycler.
5.
Sterile 1.5mL tubes
6.
Sterile work area such as biological safety cabinet or hood.
7.
Table top centrifuge with plate adapters and capacity to reach 2500 x g
8.
Vortex
Agarose Gel Electrophoresis
9.
Agarose gel electrophoresis apparatus
10.
1% agarose (molecular biology grade) TBE gel containing 0.1g/mL ethidium
bromide.
11.
Loading buffer
12.
PCR Marker suitable to cover range of 300 – 1300 bp
13.
UV transilluminator
Purification of PCR Product
14.
ExoSAP (USB® ExoSAP-IT® Cat No 78200 for 100 reactions or Illustra™
ExoProStar™ Cat No US77702 for 100 reactions)
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For In-Vitro Diagnostic Use
15.
2mM MgCl2 (Available for purchase from Conexio Genomics, product code MgCl21.0(50) or MgCl2-1.0(3000))
16.
Shaker
The use of alternative PCR purification techniques requires validation by the
user prior to use.
Sequencing Reaction
17.
BigDye® Terminator Cycle Sequencing Kit v3.1 or v1.1, Applied Biosystems™ by
Life Technologies™.
18.
5x Sequencing Reaction Buffer (Conexio Genomics, product code SEQ BUF2.0(400) or SEQ BUF-2.0(5000)) or BigDye® Terminator v3.1 or v1.1 5X
Sequencing Buffer, Applied Biosystems™ by Life Technologies™.
Purification of Sequencing Reaction Products
19.
125mM EDTA, pH8.0 (Available for purchase from Conexio Genomics, product
code EDTA-3.0(200) or EDTA-3.0(5000)).
20.
Absolute and 80% Ethanol. Each run requires freshly prepared 80% ethanol
consisting of absolute ethanol and sterile water. DO NOT USE DENATURED
ETHANOL (also known as methylated spirits in some countries).
The use of alternative sequencing purification techniques requires validation by
the user prior to use.
Denaturation and Electrophoresis of Sequencing Reaction Products
21.
Hi-Di™ Formamide, Applied Biosystems™ by Life Technologies™, product code
4311320
22.
Automated DNA Sequencer and accessories (e.g. Applied Biosystems™ by Life
Technologies™ ABI Prism® 3730), including data collection and software.
These kits have been tested and validated on the Applied Biosystems™ by Life
Technologies™ 3100, 3730 and 3730xl capillary sequencers and software.
The use of other denaturation techniques and sequencing platforms requires
validation by the user prior to use.
23.
HLA Sequencing Analysis Software (e.g. Assign™ SBT, version 3.6+ or higher
Conexio Genomics Pty Ltd).
Sample Requirements
1. Sterile water (negative/ no template control)
2. High molecular weight human genomic DNA (concentration range of 20-100ng/µL in
Tris/EDTA buffer and OD260/280> 1.8) extracted from ACD or EDTA anticoagulated
whole blood specimens. Do NOT use whole blood specimens containing heparin.
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For In-Vitro Diagnostic Use
Warnings and Safety Precautions

This kit must be used by trained and authorized laboratory personnel.

All samples, equipment and reagents must be handled in accordance with good
laboratory practice. In particular, all patient material should be considered as
potentially infectious. The use of gloves and laboratory coats is strongly
recommended. Handle and dispose of all sample material according to local and
national regulatory guidelines.

There are NO dangerous substances contained in any of the kit components.

Do NOT use reagents beyond their expiration date.

The use of kit components from different kit batches is NOT recommended. Such use
may affect the assay’s performance.

Use of reagents not included in this kit or not listed under “Materials, Reagents and
Equipment Not Supplied” (e.g. alternative Taq DNA polymerases) is NOT
recommended. Such use may affect the performance of the assay.

Care should be taken to prevent cross-contamination of DNA specimens. Change tips
between DNA specimens wherever possible.

Pre- and Post-PCR activities must be strictly physically separated. Use specifically
designated equipment, reagents and laboratory coats.

Ethidium bromide is a potential carcinogen. Protective gloves must always be used
when preparing and handling gels. Dispose of ethidium-bromide gels and buffers
according to local and national guidelines.

While viewing and photographing agarose gels under UV light, always avoid direct
exposure and use appropriate UV-blocking face protection, disposable gloves and
laboratory coats.
Symbols
The following non-standard symbols have been used:
Symbol
Description
HLA-X MIX
Locus specific PCR Mix
DNA POL – XXXX
AEX1F
DNA polymerase
HLA-A exon 1 forward sequencing primer. Refer to “Kit
Composition” and Table 4 for other sequencing primers.
Date of manufacture (required for non-EU markets).
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For In-Vitro Diagnostic Use
Procedure
1. PCR
1.1. A separate PCR reaction will need to be set up for each locus to be amplified, and for
each individual sample to be tested. Each run should include appropriate positive
control/s of known genotype, and at least one negative control for each locus being
amplified.
1.2. Prepare a fresh solution of PCR master mix each time a PCR is performed. Quickly
thaw the locus-specific PCR mix at room temperature. Once thawed, vortex briefly.
1.3. Dispense the required volume of PCR mix and DNA polymerase into a sterile tube
for the number of samples to be tested (refer to Table 1 below for the volume per
reaction). Pulse vortex the solution 3-4 times.
Locus
A
B
C
DRB1
DQB1
DPB1
Locus-specific PCR Mix 16L 16L 16L 16.7L 16.7L 16.7L
e.g. HLA-A MIX
DNA Polymerase
1L
1L
1L
0.3L
0.3L
0.3L
e.g. DNA POL – HLA-A
Table 1: Composition of the master mix required per sample.
1.4. Dispense 17L of the master mix into each reaction well.
1.5. Add 3L of sample DNA or appropriate positive control/s to each reaction well. Add
3L of sterile water to the negative control reaction well.
1.6. Seal the reaction wells. Mix gently by vortexing and centrifuge briefly.
1.7. Place the reaction wells into a thermal cycler and run according to the thermal cycling
conditions below.
95°C - 10 mins
96°C - 20 secs
60°C - 30 secs
72°C - 3 mins
33 cycles
15°C - hold
1.8. Amplification takes approximately 2.5 hours to complete.
1.9. When the PCR is completed, remove the reaction wells/plate from the thermal cycler
and either proceed directly to gel electrophoresis or store at 4°C until required.
NOTE: Purification of amplicons by ExoSAP treatment should occur within 24 hours of
completion of PCR.
2. Agarose Gel Electrophoresis
2.1. Confirm successful amplification by agarose gel electrophoresis using 2L of each
PCR product combined with 5L loading buffer (alternative volumes of loading
buffer should be validated prior to use). The use of 1% agarose gels is
recommended.
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For In-Vitro Diagnostic Use
2.2. The number and expected sizes of the resultant amplicons will vary according to the
locus and sample genotype. Expected PCR amplicon sizes are indicated in Table 2.
Locus
Expected band sizes
HLA-A
≈ 2 kbp
HLA-B
≈ 2 kbp
HLA-C
≈ 1.1 kbp and 1.4 kbp
HLA-DRB1
≈ 450 bp - 850 bp
(HH-PD5.2-5)
≈ 630 bp - 980 bp
(LG-PD5.2-7)
Banding pattern will vary depending on the
presence of specific allele groups
HLA-DQB1
HLA-DPB1
≈ 300 bp and 500 bp
(PQ-PD6.2-2)
≈ 400 bp and 500 bp
(AN-PD6.2-3)
≈ 400 bp
(HH-PD10.1)
≈400 bp, ≈780 bp and ≈1470 bp (KD-PD10.2-1)
Table 2: Expected product sizes for each assay.
3. Purification of PCR Product
NOTE: Purification systems other than ExoSAP-IT® or ExoProStarTM (e.g. Agencourt®
AMPure® XP or column-based systems) can be used to purify these PCR products. It is
strongly recommended that users validate these procedures before proceeding. If ExoSAP
treatment is to be used it is recommended that users follow the procedure described below.
3.1. Prepare a mastermix consisting of 4L of ExoSAP-IT® or ExoProStarTM and 8L of
2mM MgCl2 per sample to be purified. Gently pulse vortex to mix. Dispense 12L
of the mastermix into the reaction well of each reactive sample. Seal the wells,
vortex and then either place on a shaker or gently vortex for 2 minutes. Centrifuge
briefly before placing into the thermal cycler. Run the thermal cycler according to
the following profile:
37°C - 30 mins
80°C - 15 mins
4°C - hold
3.2. Upon completion, dilute the purified product 1:4 with sterile water. This dilution step
will ensure that there is sufficient template to perform the sequencing reactions and
ensure that the concentration of the template is sufficient to produce good quality
sequence data.
NOTE: A higher dilution factor (e.g. 1:8) may be required if consistently high signals and
associated noise and artefacts are observed. Weaker PCR products may require a lower
dilution factor.
3.3. ExoSAP treated samples may be stored at 4C until ready for use. These samples can
be stored at 4C for up to a week before use, but should be stored at -20C for long
term storage.
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For In-Vitro Diagnostic Use
4. Sequencing Reaction
NOTE: In instances where heterozygous ambiguities are to be resolved with hemizygous
sequencing primers such as HARPS®, please refer to the SBT Resolver™ HARPS®
Instructions for Use.
4.1. Table 3 lists the sequencing primers that are to be used for each locus.
HLA-A
HLA-B
HLA-C
AEX1F
AEX1R
BEX1F
BEX2F
CEX1F
CEX1R
AEX2F
AEX2R
BEX2R
BEX3F
CEX2F
CEX2R
AEX3F
AEX3R
BEX3R
BEX4F
CEX3F
CEX3R
AEX4F
AEX4R
BEX4R
CEX4F
CEX4R
CEX5F
CEX5R
CEX6F
CEX6R
CEX7F
HLA-DRB1†
HLA-DQB1
DRB1EX2F
DRB1EX2R-2
DQB1EX2F
DQB1EX2R
DRB1EX3R-2^
RB-TG344-R†
DQB1EX3F
DQB1EX3R
Or
HLA-DPB1
DPB1EX2R
DPB1EX2F
Or
Or
DRB1EX2F
DRB1EX2R-2
DQB1EX2F
DQB1EX2R-3
DPB1EX1F
DPB1EX1R
DRB1EX3F-7
DRB1EX3R-7
DQB1EX3F
DQB1EX3R
DPB1EX2F
DPB1EX2R
DPB1EX3F
DPB1EX3R
DPB1EX4F
DPB1EX4R
DPB1EX5F
DPB1EX5R
†
RB-TG344-R
PB-AG341-R*
Table 3: Sequencing primers provided for use for each locus.
RB-TG344-R is a HARP® directed to the codon 86 dimorphism. Its use is optional.
*PB-AG341-R is a HARP® directed to the codon 85 dimorphism in DPB1. Its use is also optional.
†
^DRB1EX3R-2 is a DRB1 sequencing primer in the HH-PD5.2-5 kits which behaves similar to a
HARP and is designed to sequence the following allele groups: *03, *08, *11, *12, *13, *14, *15
and *16. This primer will produce either heterozygous, hemizygous, or no sequencing data
depending on the genotype of the sample being typed. When analysing DRB1EX3R-2 data in
Assign™ against the DRB1-FullX2 reference, the resulting exon 3 data will be analysed in a
separate layer and will allow resolution of a number of allele ambiguities in exon 3, such as the
DRB1*14:01 vs *14:54 ambiguity. Its use is optional depending on the typing strategy used by the
laboratory. This is not applicable to the LG-PD5.2-7 kits as bi-directional sequencing for exon 3 is
available.
4.2. Prepare a fresh solution of sequencing primer mix on ice each time a sequence
reaction is performed. The composition and volumes for the mix indicated below are
per sample.
Page 14 of 25
For In-Vitro Diagnostic Use
Component
Sequencing primer
Volume
2µL
Sterile water
11.5µL
BigDye® Terminators
1µL
5x Seq Rxn Buffer
3.5µL
4.3. Mix each sequencing reaction mixture gently by pulse vortexing.
4.4. Dispense 18µL of the sequencing reaction mix into each appropriate reaction well.
NOTE: For runs which involve few samples with many sequencing primers, it is acceptable
to dispense the sequencing primer (2µL) directly into the individual reaction wells. A master
mix may then be created composing of sterile water, BigDye® Terminators and 5x Seq Rxn
Buffer, of which 16uL is to be dispensed into each reaction well. It is strongly recommended
that use of this alternative procedure is validated by the user prior to implementation.
4.5. Add 2µL of purified PCR product to each appropriate well.
NOTE: Care must be taken to prevent cross-contamination of sequence reactions.
4.6. Seal the reaction wells, mix gently and centrifuge briefly to ensure that the contents
are located at the base of each reaction well.
4.7. Place the reaction wells into a thermal cycler and run according to the following
profile:
Number of cycles
Temperature and time
25
96°C - 10 sec
50°C - 5 sec
60°C - 2 min
1
4°C - hold
4.8. Once the program is complete, remove the reaction wells/plate from the thermal
cycler and either proceed directly to purification of the reaction products or store in
the dark at 4C until required. It is recommended that samples are purified and run
on the DNA sequencer within 24 hours.
5. Purification of Sequencing Reaction Products
NOTE: Purification of the reaction products may be carried out by procedures other than the
ethanol precipitation method described here. It is strongly recommended that users validate
these procedures before proceeding.
5.1. Briefly centrifuge the reaction wells/plates before proceeding. If reusable lids/caps
have been used during thermal cycling, label the lids/caps to avoid crosscontamination.
5.2. Carefully remove the seals.
5.3. To each reaction well add 5µL of 125mM EDTA, pH8.0. Ensure that the EDTA
reaches the base of the reaction well.
5.4. Add 60µL of 100% ethanol to each reaction well. Seal the wells/plate and vortex
briefly but thoroughly to ensure thorough mixing.
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For In-Vitro Diagnostic Use
5.5. Pellet the extension products by centrifuging at 2000g for 45 minutes.
IMMEDIATELY PROCEED TO THE NEXT STEP. If this is not possible, recentrifuge for an additional 10 minutes before proceeding.
5.6. Remove the seals to the reaction wells and discard the supernatant by inverting the
reaction wells onto paper towel or tissues.
5.7. Place the inverted reaction wells and paper towel or tissue into the centrifuge.
Centrifuge at 350g for 1 minute to remove any residual supernatant.
5.8. Remove the reaction wells from the centrifuge and place in an upright position on the
work bench. Discard the paper towel or tissues.
5.9. Prepare fresh solution of 80% ethanol with absolute ethanol and sterile water.
5.10. Add 60µL of 80% ethanol to each well. Reseal the wells and vortex briefly.
5.11. Spin at 2000g for 5 minutes.
5.12. Repeat steps 5.6 and 5.7.
5.13. Remove the reaction wells from the centrifuge and discard the paper towel. Reseal
the reaction wells and proceed to the denaturation step. Otherwise store at -20C in
the dark. It is recommended that the extension products are run on the DNA
sequencer within 24 hours of setting up the sequencing reactions.
6. Denaturation & Electrophoresis of Sequencing Reaction Products
NOTE: The procedure for the denaturation of extension products in Hi-Di™ Formamide
described here may not be necessary if purification procedures other than the ethanol
precipitation have been used. It is strongly recommended that users validate alternative
procedures before proceeding.
6.1. Add 12µL of Hi-Di™ Formamide to each reaction well. Vortex and centrifuge the
wells/plate briefly.
6.2. Incubate the reaction wells at 98C for 5 minutes. Following incubation, ensure that
the reaction wells are cooled quickly to room temperature (e.g. place on ice or use
the thermal cycler to perform the denaturation and cooling steps) before being placed
on the sequencer. If it is not possible to run the plates immediately, store at 4C until
required.
NOTE: Ensure that there are no air bubbles in the reaction wells. These can enter and damage
the capillary.
6.3. Load the reaction wells/plate onto the automated sequencer and prepare the data
collection file according to the sequencer manufacturer specifications.
6.4. The following instrument parameters have been validated by the manufacturer using
Big Dye® Terminator Sequencing Kit v3.1 and POP-7™. These parameters may
require user validation for other polymers, sequencing chemistries and instruments.
Please refer to the appropriate instrument user’s manual for detailed instructions and
guidance (e.g. ensure that the dye set setting is appropriate for the chemistry used,
for example v1.1 Big Dye® Terminator sequencing chemistry will require a different
dye set).
Parameter
Setting
Dye set
Z_BigDyeV3
Page 16 of 25
For In-Vitro Diagnostic Use
Mobility file
KB_3730_POP7_BDTV3
Basecaller
KB.bcp
Run Module
Regular FastSeq50_POP7
Injection time
15 sec
Run time
3000 sec
6.5. Use the instrument’s data collection software to process the raw collected data and
create the sequence files. Please refer to the appropriate instrument user’s manual for
detailed instructions and guidance.
7. Editing and analysis of electropherograms
The SBT Resolver™ kits were developed and validated using the Assign™ SBT and
Assign™ ATF software developed by Conexio Genomics Pty Ltd. Users are recommended
to use Assign SBT versions 3.6+ and higher as these versions of the software utilise setting
and reference files specifically designed for the SBT Resolver™ typing kits and HARPS®.
For more details in relation to the operation of these software please refer to the applicable
user manuals available for download on the Conexio Genomics website (http://www.conexiogenomics.com).
The sequencing based typing data generated using the SBT Resolver™ typing kits should be
analysed against the following Assign™ reference files which are provided by Conexio
Genomics:
Assay
Product Code
Assign Reference File
SBT Resolver™ HLA-A
XH-PD1.1-2
A.xml
SBT Resolver™ HLA-B
BS-PD2.1-2
B.xml
SBT Resolver™ HLA-C
HH-PD3.2-2
C.xml or Cw.xml
SBT Resolver™ HLA-DRB1
HH-PD5.2-5
DRB1-FullX2.xml
LG-PD5.2-7
527_DRB1.xml
PQ-PD6.2-2
DQB1.xml
AN-PD6.2-3
623_DQB1.xml
HH-PD10.1
DPB1.xml
KD-PD10.2-1
DPB1.xml
SBT Resolver™ HLA-DQB1
SBT Resolver™ HLA-DPB1
Page 17 of 25
For In-Vitro Diagnostic Use
Performance Characteristics
Accuracy
Panels of up to 93 samples from the UCLA International DNA Exchange proficiency testing
program (2008 - 2010) used for internal testing for the SBT Resolver™ kits yielded the
following results:
Locus
Number
of
samples
tested
Diagnostic Diagnostic
sensitivity specificity
(% of
successful
PCRs)
(% of
genotypes
obtained)
Number
Number of
of
heterozygous
discordant
samples
samples
Number
of
unique
alleles
HLA-A
81
100%
100%
0
74
20
HLA-B
82
100%
98.8%
0
79
81
HLA-C
39
97.5%
97.5%
0
35
21
HLA-DRB1
93
96.7%
96.7%
0
84
39
HLA-DQB1
42
100%
100%
0
36
14
38
100%
100%
0
34
15
77
100%
100%
0
60
18
16
100%
100%
2*
14
13
(PQ-PD6.2-2)
HLA-DQB1
(AN-PD6.2-3)
HLA-DPB1
(HH-PD10.1)
HLA-DPB1
(KD-PD10.2-1)
* The two discordant samples contained additional sequence information outside exon 2 that
was not reported by the UCLA International DNA Exchange proficiency testing program.
One sample contained 131:01, but was reported as 13:01 by the UCLA International DNA
Exchange proficiency testing program. The alleles differ in exons 3 and 4. The other sample
contained 107:01, but was reported as 13:01 by the UCLA International DNA Exchange
proficiency testing program. These alleles differ in exon 1.
For the SBT Resolver HLA-DRB1 kits (product code LG-PD5.2-7), a panel of 23 well
characterised samples, covering a broad range of alleles was used for internal testing. In
addition, a panel of 293 externally sourced samples were also typed without a priori
knowledge of other HLA typing data. These samples were also tested with the SBT
Resolver™ HLA-DQB1 assay (PQ-PD6.2-2). In those cases where a homozygous result was
obtained, the DQB1/ DRB1 associations for those samples were examined to confirm the
result as well as to detect instances where allele-drop out may have occurred.
The testing yielded the following results:
Locus
Number
of
samples
tested
Diagnostic Diagnostic
sensitivity specificity
HLA-DRB1
23
100%
100%
0
23
12
286*
97.9%
99.6%
0
253
33
Page 18 of 25
Number
Number of
Number
of
heterozygous of unique
discordant
samples
alleles
samples
For In-Vitro Diagnostic Use
* Six samples failed to amplify due to poor quality DNA samples. One sample was found to
contain contaminating DNA, the source of which occurred at the laboratory from which the
samples were obtained. As a result of the contamination, a genotype could not be obtained for
that sample.
Sequence analysis of PCR and sequencing primer sites and performance evaluation studies
have not identified any common and well documented alleles that have not been amplified
through the recommended use of these kits. For further information refer to the SBT
Resolver™ Primer Analysis document available with each Assign™ SBT reference release,
downloadable from the Conexio Genomics website (http://www.conexio-genomics.com).
Detection Limit
The recommended concentration of high molecular weight human genomic DNA is 20100ng/L. Internal testing has shown that samples with concentrations as low as 5ng/L can
also be used. Correct genotypes were also obtained from poor quality or sheared DNA.
Specificity
Conexio Genomics Pty Ltd’s SBT Resolver™ kits are locus specific assays. Use of the kits
according to these instructions should only amplify a single locus. In most instances the use
of the sequencing primers incorporated in each kit will produce a HLA typing for most
samples without the need for further resolution. In those instances where heterozygous
ambiguities remain, the use of resolving sequencing primers (such as SBT Resolver™
HARPS®) is recommended.
It should be noted that mutations at amplification or sequencing primer sites are possible and
may result in allele drop-out. Samples that suggest a homozygous typing result must be
confirmed by alternative procedures.
Limitations and Cautions

It is strongly recommended that these kits are validated by the user prior to
implementation in the laboratory using samples whose HLA type has been determined by
other molecular based procedures. In particular, any deviations from this procedure (e.g.
the use of alternative PCR or DNA sequencing purification procedures) must be validated
by the user prior to implementation.

These kits have been validated using panels of samples whose genotypes cover a broad
range of alleles. However it should be noted that rare alleles and alleles with
polymorphisms in amplification and sequencing primer sites may be encountered and
these may not be amplified or sequenced.

The nature of HLA sequence based typing is such that factors other than the PCR mix
may result in preferential amplification or allele drop out. As a consequence, apparent
homozygous typing results should be confirmed using alternative methods and/or family
genotyping.

A positive control (human DNA) and negative control (sterile water) must be included on
every PCR run. The positive control must produce a PCR product of the appropriate size
depending on the locus amplified and the resultant sequence must be in concordance with
the sample’s genotype. There must be no PCR products in the negative template control
for each experiment. If a band is evident contamination may have occurred at some level
and the run must be repeated.

Occasionally there may be additional, fainter PCR products evident. These additional
bands do not interfere with sequence results or quality.
Page 19 of 25
For In-Vitro Diagnostic Use
License
The SBT Resolver™ kits contain GoTaq® Hot Start Polymerase (DNA POL) which is
manufactured by Promega Corporation for distribution by Conexio Genomics Pty Ltd.
Licensed to Promega under U.S. Patent Nos. 5,338,671 and 5,587,287 and their
corresponding foreign patents.
Bibliography
1. Sayer D, Whidborne R, Brestovac B, Trimboli F, Witt C, Christiansen F (2001): HLADRB1 DNA sequencing based typing: an approach suitable for high throughput typing
including unrelated bone marrow registry donors. Tissue Antigens 57: 46-54.
2. Sayer D, Whidborne R, DeSantis D, Rozemuller EH, Christiansen F, Tilanus MG (2004).
A multicentre international evaluation of single-tube amplification protocols for
sequencing-based typing of HLA-DRB1 and HLA-DRB3, 4, 5. Tissue Antigens 63: 412423.
3. Assign™ SBT v3.6+ Operator Manual, Conexio Genomics Pty Ltd
4. Assign™ SBT v4.7 Operator Manual, Conexio Genomics Pty Ltd
5. Assign™ SBT v471 Operator Manual, Conexio Genomics Pty Ltd
6. More information regarding the UCLA DNA Exchange Program can be found at:
http://www.hla.ucla.edu/cellDNA/DNA/programInfo.htm.
7. Current HLA alleles can be found at http://www.ebi.ac.uk/imgt/hla.
Page 20 of 25
For In-Vitro Diagnostic Use
Troubleshooting
Problem
Possible cause(s)
Solution
No or weak PCR product
Poor quality DNA
Assess DNA quality by gel
electrophoresis. Intact DNA
should be approx 3kb with
little or no evidence of
smearing on gel. Re-extract
DNA and repeat PCR where
possible.
Check concentration of DNA
is between 20-100ng/L. Reextract DNA and repeat PCR
where possible.
Avoid the use of whole blood
specimens containing heparin.
Re-extract DNA and repeat
PCR where possible.
Repeat
PCR.
Ensure
mastermix components are
added and mixed sufficiently
by vortexing.
Check the thermal cycling run
parameters.
Check the run history to ensure
that the run was not terminated
prematurely.
Ensure that the thermal cycler
is operating according to
manufacturer’s specifications
and is regularly maintained.
Submerge the gel in a staining
bath containing 1X TBE with
0.5mg/mL ethidium bromide.
Destain in 1X TBE before
taking gel image.
Ensure ethidium bromide is
added to gel prior to pouring.
Wherever possible use sterile
water with a neutral pH.
Insufficient quantity of DNA
added to PCR.
Presence of PCR inhibitors in
genomic DNA
DNA polymerase not added to
the mastermix or insufficient
mixing of mastermix prior to
addition to samples.
Thermal cycling problems
No ethidium bromide added to
the gel.
No or weak PCR product
for the exon 3-5 band for
the KD-PD10.2-1 assay
Incorrect band sizes
DNA samples are eluted or
diluted in water that can have a
slightly acidic pH.
Poor quality DNA
Amplification of samples of
very poor quality may result in
weak amplification of the exon
3-5 amplicon. Typing can still
be achieved using exon 1 and
2 sequence data. Alternatively
re-extract DNA and repeat
PCR where possible.
Incorrect kit used
Check that the appropriate kit
is used.
Incorrect
thermal
cycling Check the thermal cycle
program used.
parameters.
Page 21 of 25
For In-Vitro Diagnostic Use
PCR contamination
Weak signal intensity of
electropherograms
Weak PCR product
Insufficient reaction products
applied to sequencer
Problems during purification of
sequencer products
Signal intensity is too
high (Presence of high
fluorescent peaks –
artefacts)
Too much PCR product
Too much reaction products
applied to sequencer.
Noisy baseline (high
background)
Contaminated PCR product
Amplification of closely related
HLA genes
Poor PCR purification
Contaminated
reactions
sequencing
Page 22 of 25
Check the negative control for
evidence of contamination.
Decontaminate work area and
repeat PCR.
Repeat PCR to identify source
of contamination. Consider
using a fresh kit.
If the genomic DNA of a
sample
appears
to
be
contaminated, re-extract or
obtain an alternative source of
DNA.
Check gel image. Sequencing
weak PCR bands is NOT
recommended as the sequence
quality may be insufficient for
SBT.
Consider using a lower
dilution factor (e.g. 1:2, 1:3)
after PCR purification.
Check sequencer parameters.
Injection time and voltage may
need to be increased.
Use extreme care when
discarding the supernatant as it
may dislodge the pellet.
Check the gel image. Consider
using a higher dilution factor
following PCR purification.
Check the amount of DNA
polymerase used in the PCR.
Check instrument parameters.
Consider
reducing
the
injection time and voltage.
Refer to corrective actions
listed above.
Check
thermal
cycling
parameters.
Ensure ExoSAP treatment is
undertaken according to kit’s
user instructions.
Ensure that the PCR mixture is
mixed
thoroughly
with
ExoSAP
Consider
using
ExoSAP
following the manufacturers
procedure (increasing the
amount of enzyme), or
consider
an
alternative
purification technique.
Ensure that all steps are taken
to
prevent
cross
contamination. Change pipette
tips wherever possible. Add
liquids at the top of the
reaction
wells.
Prevent
For In-Vitro Diagnostic Use
aerosols.
Contaminated sequencing
primer
Presence of Dye blobs
Check sequence quality of the
other sequencing primers and
other samples using the same
primer.
Consider using a fresh aliquot
of sequencing primer.
Contaminated dye terminator Repeat sequencing with fresh
mix or sequencing buffer
aliquot of reagents.
Poor purification of sequencing Repeat sequencing and ensure
products.
that purification is undertaken
according to manufacturer’s
instructions.
Poor purification of sequencing Purify products according to
products
kit instructions.
Ensure products are washed
sufficiently with 80% ethanol.
Page 23 of 25
For In-Vitro Diagnostic Use
Related Products
CE marked IVDs:
Product code: CGX0036+
Product code: CGX00470
Product code: CGX00471
Product codes:
C1-TT98-F(20)
C1-AC98-F(20)
C1-TC98-F(20)
C1-TA98-F(20)
C1-CA102-F(20)
C1-CT102-F(20)
C1-CC102-F(20)
C1-AG203-F(20)
C1-GT240-F(20)
C1-TT368-F(20)
C1-GG307-R(20)
C1-GG363-AF(20)
C1-TA363-F(20)
C1-AT362-F(20)
C1-AC497-F(20)
C1-TA368-F(20)
C1-GT355-R(20)
C1-GG362-R(20)
C1-CT423-F(20)
C1-CG570-R(20)
C1-BTA-F(20)
C1-BCG-F(20)
C1-CC144-F(20)
C1-AC206-F(20)
C1-GC209-F(20)
C1-GA206-F(20)
C1-CG319-F(20)
C1-CA309-R(20)
C1-GAT309-R(20)
C1-GAA309-R(20)
C1-AG360-F(20)
C1-GC363-F(20)
C1-GG363-BF(20)
C1-TA420-F(20)
C1-AC362-F(20)
C1-CC486-F(20)
C1-CT559-R(20)
C1-GA559-R(20)
C1-AC559-R(20)
C1-GG572-R(20)
C1-CG572-R(20)
C1-GAG601-R(20)
C1-CT97-F(20)
C1-CT112-F(20)
C1-CG134-F(20)
C1-AG270-F(20)
C1-AC302-R(20)
C1-GC302-R(20)
C1-CG343-F(20)
C1-CA343-F(20)
C1-GA361-F(20)
C1-TG539-R(20)
C1-GG539-R(20)
C1-AA601-R(20)
C1-AG595-R(20)
RB-01-F(20)
RB-04-F(20)
RB-09-F(20)
RB-15-F(20)
RB-52-F(20)
RB-GG125-F(20)
RB-AA197-F(20)
RB-TT197-F(20)
RB-GT196-F(20)
RB-GA196-F(20)
RB-TA164-F(20)
RB-TT227-F(20)
RB-AT258-F(20)
RB-GC258-F(20)
RB-CT257-R(20)
RB-AT257-R(20)
RB-TT321-R(20)
RB-GT344-R(20)
RB-TG344-R(20)
QB-TA122-F(20)
QB-CT173-F(20)
QB-TA185-F(20)
QB-CG353-R(20)
QB-GG353-R(20)
PB-AT251-R(20)
PB-GT313-R(20)
PB-TAC121-F(20)
PB-GG341-R(20)
PB-GC194-F(20)
PB-AG341-R(20)
PB-GC112-F(20)
QB-TA173-F(20)
For Research Use Only:
AN-PD11.0-0(20)
AN-PD11.0-0(50)
SBT Resolver™ HLA-DRB3 kit (20 and 50 tests)
AN-PD12.0-0(20)
AN-PD12.0-0(50)
SBT Resolver™ HLA-DRB4 kit (20 and 50 tests)
AN-PD13.0-0(20)
AN-PD13.0-0(50)
SBT Resolver™ HLA-DRB5 kit (20 and 50 tests)
LC-PD2.9(20)
LC-PD2.9(50)
SBT Resolver™ HLA-B57 kit (20 and 50 tests)
Note: the products listed above are licensed as IVDs in Australia
General Purpose Laboratory Reagents
Page 24 of 25
For In-Vitro Diagnostic Use
MgCl2 – 1.0(50)
MgCl2 - 1.0(3000))
2mM MgCl2
SEQ BUF – 2.0(400)
SEQ BUF – 2.0(5000)
5x Seq Rxn Buffer
EDTA – 3.0(200)
EDTA – 3.0(5000)
125mM EDTA, pH8.0
Please contact your local distributor for further details.
0197
Self-certified kits:
HH-PD3.2-2(20)
HH-PD3.2-2(50)
PQ-PD6.2-2(20)
PQ-PD6.2-2(50)
AN-PD6.2-3(20)
AN-PD6.2-3(50)
HH-PD10.1(20)
HH-PD10.1(50)
KD-PD10.2-1(20)
KD-PD10.2-1(50)
SBT Resolver™ HLA-C kit (20 and 50 tests)
SBT Resolver™ HLA-DQB1 kit (20 and 50 tests)
SBT Resolver™ HLA-DPB1 kit (20 and 50 tests)
Support and Contact Details
Conexio Genomics Pty Ltd
PO Box 1294
Fremantle 6959
Western Australia
Australia
Tel: +61-08-9336-4212
email: support@conexio-genomics.com
Skype: conexiocgx
Website: www.conexio-genomics.com
Or your local distributor
For ordering details, please refer to the Olerup website (http://www.olerup.com).
Conexio and HARPS are trademarks of Conexio 4 Pty Ltd. HARPS® is a registered trademark
in some territories.
Page 25 of 25
For In-Vitro Diagnostic Use
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