Instructions for Use

Instructions for Use
Instructions for Use
PCR Amplification and Sequencing of the HLA-DRB3,-DRB4 and
-DRB5 Loci
Version No: 3.0
Issue Date: March 2014
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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Contents
PRINCIPLE ........................................................................................................................................ 3
INTENDED USE ................................................................................................................................ 3
KIT COMPOSITION ......................................................................................................................... 4
STORAGE REQUIREMENTS .......................................................................................................... 5
MATERIALS, REAGENTS AND EQUIPMENT NOT SUPPLIED ............................................... 5
SAMPLE REQUIREMENTS ............................................................................................................ 6
WARNINGS AND SAFETY PRECAUTIONS ................................................................................. 7
SYMBOLS .......................................................................................................................................... 7
PROCEDURE ..................................................................................................................................... 8
1.
2.
3.
4.
5.
6.
7.
PCR ........................................................................................................................................ 8
AGAROSE GEL ELECTROPHORESIS ........................................................................................... 8
PURIFICATION OF PCR PRODUCT ............................................................................................. 9
SEQUENCING REACTION .......................................................................................................... 9
PURIFICATION OF SEQUENCING REACTION PRODUCTS ............................................................ 10
DENATURATION & ELECTROPHORESIS OF SEQUENCING REACTION PRODUCTS ....................... 11
EDITING AND ANALYSIS OF ELECTROPHEROGRAMS ................................................................ 12
PERFORMANCE CHARACTERISTICS ...................................................................................... 13
ACCURACY ..................................................................................................................................... 13
DETECTION LIMIT ........................................................................................................................... 13
SPECIFICITY .................................................................................................................................... 13
LIMITATIONS AND CAUTIONS .................................................................................................. 13
LICENSE .......................................................................................................................................... 14
TROUBLESHOOTING ................................................................................................................... 15
RELATED PRODUCTS .................................................................................................................. 17
SUPPORT AND CONTACT DETAILS .......................................................................................... 19
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For In-Vitro Diagnostic Use
Principle
The HLA Sequencing Based Typing (SBT) procedure described here, involves the initial
locus specific PCR amplification of the HLA-DRB3, -DRB4 and -DRB5 genes. Presence of
target and the internal control amplicons are visualised by agarose gel electrophoresis.
Samples that are positive for the HLA-DRB3, -DRB4 or -DRB5 genes will produce two
amplicons (the target amplicon and internal control) while negative samples will produce a
single amplicon only (internal control). Positive samples are then further characterised by
DNA sequencing following treatment with ExoSAP-IT® to remove unincorporated primers
and dNTPs. The target amplicon is then used as a template for direct automated fluorescent
DNA sequencing using customized sequencing primers and the BigDye® 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 analysed with Assign™ SBT sequence analysis
software from Conexio Genomics Pty Ltd.
Intended Use
These Conexio Genomics’ SBT Resolver™ SBT kits are used for the typing of the HLA
Class II genes, HLA-DRB3, -DRB4 and -DRB5 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 kits are not to be 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)
Locus
HLA-DRB3
AN-PD11.0-0(20)
AN-PD11.0-0(50)
HLA-DRB4
AN-PD12.0-0(20)
AN-PD12.0-0(50)
HLA-DRB5
AN-PD13.0-0(20)
AN-PD13.0-0(50)
20 tests
50 tests
20 tests
50 tests
20 tests
50 tests
DNA POL – DRB3
1 x 10L
HLA-DRB3 MIX
1 x 370L
DNA POL – DRB3
1 x 20L
HLA-DRB3 MIX
1 x 920L
DNA POL – DRB4
1 x 10L
HLA-DRB4 MIX
1 x 370L
DNA POL – DRB4
1 x 20L
HLA-DRB4 MIX
1 x 920L
DNA POL – DRB5
1 x 10L
HLA-DRB5 MIX
1 x 370L
DNA POL – DRB5
1 x 20L
HLA-DRB5 MIX
1 x 920L
†
The PRE-PCR component of each kit consists of a vialof a locus-specific PCR mix (e.g.
MgCl2, locus specific PCR primers, and a single vial of DNA polymerase (e.g.
The POST-PCR kit contains sequencing primers (e.g.
DRB3EX2F
DRB3EX2F
DRB3EX2R
1 x 44L each
DRB3EX2F
DRB3EX2R
1 x 110L each
DRB4EX2F
DRB4EX2R
1 x 44L each
DRB4EX2F
DRB4EX2R
1 x 110L each
DRB5EX2F
DRB5EX2R
1 x 44uL each
DRB5EX3F
DRB5EX2F
DPB5EX2R
1 x 110uL each
DRB5EX3F
HLA-DRB3 MIX
DNA POL – DRB3
) consisting of PCR buffer, dNTPs,
).
).
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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 these kits have indicated a shelf life of two and a half years
from the 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 tested using the following thermal cyclers:
MJ Research PTC 225 DNA Engine DYAD™, Applied Biosystems™ by Life
Technologies™ GeneAmp® 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 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-IT (USB Products Cat No 78200 for 100 reactions)
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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 Ethanol. Each run needs freshly prepared 80% ethanol solution consisting of
absolute ethanol and sterile water. DO NOT USE DENATURED ETHANOL.
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 (eg 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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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 biological 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 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
HLA-X MIX
DNA POL – XXXX
DRB3EX2F
Description
Locus specific PCR Mix
DNA polymerase
HLA-DRB3 exon 3 forward sequencing primer. Refer to “Kit
Composition” and Table 3 for other sequencing primers.
Date of manufacture (required for non-EU markets).
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Procedure
1. PCR
1.1. Set up one reaction for each sample, for each locus being amplified. Include
appropriate positive and negative amplification controls of known genotype and at
least one no template control for each group of samples being amplified.
1.2. Prepare a fresh solution of PCR master mix each time a PCR is performed. Quickly
thaw the required number of vials of the appropriate PCR Mix. Once thawed vortex
briefly.
1.3. Dispense the required amount of PCR mix and DNA polymerase into a sterile tube
for the number of samples to be tested. Refer to Table 1 below. Pulse vortex the
solution 3-4 times.
Locus
DRB3
Locus-specific PCR Mix 16.7L
e.g.
HLA-DRB3 MIX
DNA Polymerase
e.g.
DNA POL-DRB3
0.3L
.
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 control sample to each reaction well. Add
3L of sterile water to the no template 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 amplify the target sequence
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 plate from the thermal cycler and either
proceed directly to gel electrophoresis or store at 4°C until required.
NOTE: Purification of positive amplicons by ExoSAP-IT® treatment should occur within 24
hours of completion of PCR.
2. Agarose Gel Electrophoresis
2.1. Confirm successful amplification of the internal control amplicon in for all DNA
samples tested, and the applicable HLA-DRB3, -DRB4 and -DRB5 target amplicons
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in positive control and positive DNA samples by agarose gel electrophoresis, using
5L of each PCR product combined with 5L of loading buffer (alternative volumes
of loading buffer should be validated prior to use). The use of 1% agarose gels is
recommended.
2.2. All samples tested using the HLA-DRB3, -DRB4 and -DRB5 PCR mixes should
amplify the internal control amplicon regardless of the HLA-DRB3, -DRB4, -DRB5
genotype. Positive samples should amplify both the internal control amplicon plus
the target amplicon. The expected sizes of each amplicon are listed in Table 2.
Locus
Expected band sizes
HLA-DRB3 target amplicon
≈ 640 bp
HLA-DRB4 target amplicon
≈ 460 bp
HLA-DRB5 target amplicon
≈ 470 bp
Internal control band
≈ 400 bp
Table 2: Expected product sizes for each assay.
3. Purification of PCR Product
NOTE: Purification systems other than ExoSAP-IT® (e.g. ExoSTAR™, 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-IT®
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® and 8L of 2mM MgCl2 per
sample. Gently pulse vortex to mix. Dispense 12L of the mastermix into the
reaction well of each positive sample. Seal the tubes, vortex, and place on a shaker or
gently vortex for 2 mins. Centrifuge briefly before placing into the thermal cycler.
Run the thermal cycler according to the following profile:
37°C - 30mins
80°C - 15mins
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-IT® treated samples may be stored at 4C until ready for use. ExoSAP-IT®
treated 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.
4. Sequencing Reaction
NOTE: Only HLA-DRB3, -DRB4 and -DRB5 positive samples identified by gel
electrophoresis should be sequenced using the following procedure.
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4.1. Table 3 lists the sequencing primers that are to be used for each locus.
Locus
Sequencing Primers
HLA-DRB3
DRB3EX2F
DRB3EX2R
HLA-DRB4
DRB4EX2F
DRB4EX2R
HLA-DRB5
DRB5EX2F
DRB5EX2R
DRB5EX3F
Table 3: Sequencing primers provided to sequence the positive samples for each locus.
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 are indicated per
sample.
Component
Sequencing primer
Volume
2 µL
Sterile water
11.5 µL
®
BigDye Terminators
1 µL
5X Sequencing buffer
3.5 µL
4.3. Mix each sequencing reaction mix gently by pulse vortexing.
4.4. Dispense 18µL of the sequencing reaction mix to each appropriate reaction tube/well.
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 tubes, mix gently and centrifuge briefly to ensure that the contents
are located at the base of each reaction tube.
4.7. Place the reaction tubes into a thermal cycler and run according to the following
profile:
Number of cycles
Temperature and time
25
96°C – 10sec
50°C – 5sec
60°C – 2min
1
4°C - hold
4.8. Once the program is complete, remove the reaction tubes from the thermal cycler and
either proceed directly to purification of the reaction products or store 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.
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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 seal.
5.3. To each reaction tube add 5µL of 125mM EDTA, pH8.0. Ensure that the EDTA
reaches the base of the reaction tube.
5.4. Add 60 µL of 100% ethanol to each reaction well. Seal the plate and vortex briefly
but thoroughly to ensure thorough mixing.
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 tubes and discard the supernatant by inverting the
reaction tubes onto paper towel or tissues.
5.7. Place the inverted reaction tubes and paper towel or tissue into the centrifuge.
Centrifuge at 350g for 1 minute to remove any residual supernatant.
5.8. Remove the reaction tubes from the centrifuge and replace in an upright position on
the work bench. Discard the paper towel or tissues.
5.9. Prepare a fresh solution of 80% ethanol with absolute ethanol and sterile water.
5.10. Add 60µL of 80% ethanol to each reaction tube/well. Reseal the tubes and mix by
vortexing briefly.
5.11. Spin at 2000g for 5 minutes.
5.12. Repeat steps 5.6 to 5.7.
5.13. Remove the reaction tubes from the centrifuge and discard the paper towel. Reseal
the reaction tubes 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 tube. Vortex and centrifuge the
tubes briefly.
6.2. Incubate the reaction tubes 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.
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6.3. Load the reaction 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
Mobility file
KB_3730_POP7_BDTV3
Basecaller
KB.bcp
Run Module
Regular FastSeq50_POP7
Injection time
15 sec
Collection 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 3.6+ or 4.7 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-DRB3
AN-PD11.0-0
DRB3.xml
SBT Resolver™ HLA-DRB4
AN-PD12.0-0
DRB4.xml
SBT Resolver™ HLA-DRB5
AN-PD13.0-0
DRB5.xml
Alternatively, the sequencing data for all of the assays can be analysed against the D345.xml
reference so long as the naming convention is such that each locus is analysed separately.
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Performance Characteristics
Accuracy
Panels of up to 57 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
Number of
samples
containing
target locus
Number of
mistyped
samples
Number of
heterozygous
samples
Number of
unique
alleles
HLA-DRB3
54
22
0
0
3
HLA-DRB4
56
33
0
1
3
HLA-DRB5
57
15
0
0
4
Sequence analysis of PCR and sequencing primer sites and performance evaluation has not
identified any common and well documented alleles that are not amplified through the
recommended use of these kits.
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.
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.
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
A positive control (human DNA sample known to have HLA-DRB3/DRB4/DRB5), a
negative control (human DNA sample known to be negative for HLADRB3/DRB4/DRB5) and no template control (sterile water) must be included on every
PCR run. The positive control must produce two amplicons of the appropriate size and the
resultant sequence must be in concordance with the sample’s genotype. The negative
control must produce a single internal control amplicon of the appropriate size. There
must be no PCR products in the no 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 larger, fainter PCR products evident. These additional bands
do not interfere with sequence results or quality.
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.
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Troubleshooting
Problem
Possible cause(s)
No or weak PCR product
Poor quality DNA
Incorrect band sizes
Solution
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.
Insufficient quantity of DNA Check concentration of DNA
added to PCR.
is between 20-100ng/L. Reextract DNA and repeat PCR
where possible.
Presence of PCR inhibitors in Avoid the use of whole blood
genomic DNA
specimens containing heparin.
Re-extract DNA and repeat
PCR where possible.
DNA polymerase not added to Repeat
PCR.
Ensure
the mastermix or insufficient mastermix components are
mixing of mastermix prior to added and mixed sufficiently
addition to samples.
by vortexing.
Thermal cycling problems
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.
No ethidium bromide added to Submerge the gel in a staining
the gel.
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.
DNA samples are eluted or Wherever possible use sterile
diluted in water that can have a water with a neutral pH.
slightly acidic pH.
Incorrect kit used
Check that the appropriate kit
has been used.
Incorrect
thermal
cycling Check the thermal cycle
program used.
parameters.
PCR contamination
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.
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Problem
Weak signal intensity of
electropherograms
Possible cause(s)
Solution
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 16 of 19
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 (eg 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-IT® treatment
is undertaken according to
kit’s user instructions.
Ensure that the PCR mixture is
mixed
thoroughly
with
ExoSAP-IT®.
Consider using Exo-SAP-IT®
following the manufacturer’s
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
aerosols.
For In-Vitro Diagnostic Use
Problem
Possible cause(s)
Solution
Contaminated sequencing
primer
Check sequence quality of the
other sequencing primers and
other samples using the same
primer.
Consider using a fresh aliquot
of sequencing primer.
Repeat sequencing with fresh
aliquot of reagents.
Repeat sequencing and ensure
that purification is undertaken
according to manufacturer’s
instructions.
Purify products according to
kit
instructions.
Ensure
products
are
washed
sufficiently with 80% ethanol.
Contaminated dye terminator
mix or sequencing buffer
Poor purification of sequencing
products.
Presence of Dye blobs
Poor purification of sequencing
products
Related Products
CE marked IVDs:
XH-PD1.1-2(20)
XH-PD1.1-2(50)
SBT Resolver™ HLA-A kit (20 and 50 tests)
BS-PD2.1-2(20)
BS-PD2.1-2(50)
SBT Resolver™ HLA-B kit (20 and 50 tests)
HH-PD5.2-5(20)
HH-PD5.2-5(50)
SBT Resolver™ HLA-DRB1 kit (20 and 50 tests)
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)
Page 17 of 19
For In-Vitro Diagnostic Use
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)
Self-certified SBT Resolver™ typing kits:
HH-PD3.2-2(20)
HH-PD3.2-2(50)
SBT Resolver™ HLA-C kit (20 and 50 tests)
PQ-PD6.2-2(20)
PQ-PD6.2-2(50)
SBT Resolver™ HLA-DQB1 kit (20 and 50 tests)
HH-PD10.1(20)
HH-PD10.1(50)
SBT Resolver™ HLA-DPB1 kit (20 and 50 tests)
Self-certified HARPS:
QB-TA122-F(20)
QB-TA173-F(20)
QB-CT173-F(20)
QB-TA185-F(20)
QB-CG353-R(20)
PB-GC112-F(20)
PB-TAC121-F(20)
PB-GC194-F(20)
PB-AT251-R(20)
PB-GT313-R(20)
PB-AG341-R(20)
PB-GG341-R(20)
QB-GG353-R(20)
Assign™ SBT software (Self-certified):
Product code: CGX0036+
Product code: CGX00470
0197
For Research Use Only:
LC-PD2.9(20)
LC-PD2.9(50)
SBT Resolver™ HLA-B57 kit (20 and 50 tests)
General Purpose Laboratory Reagents
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.
Page 18 of 19
For In-Vitro Diagnostic Use
Support and Contact Details
Conexio Genomics Pty Ltd
2/49 Buckingham Dr
Wangara 6065
Western Australia
Tel: +61-422-863-227
email: support@conexio-genomics.com
Skype: conexiocgx
Website: http://www.conexio-genomics.com
Or your local distributor.
Conexio and HARPS are trademarks of Conexio 4 Pty Ltd. HARPS® is a registered trademark
in some territories.
Page 19 of 19
For In-Vitro Diagnostic Use
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