SBT Resolver B57 Kit IFU

SBT Resolver B57 Kit IFU
Instructions for Use
PCR Amplification and Sequencing of the DRB3/DRB4/DRB5
Version No: 1.0
Issue Date: February 2012
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.
Conexio Genomics Pty Ltd
8/31 Pakenham St
Fremantle 6160
Western Australia
Australia
Page 1 of 15
For Research Use Only
Contents
PRINCIPLE ............................................................................................................................................ 3
KIT COMPOSITION ............................................................................................................................ 4
STORAGE REQUIREMENTS ............................................................................................................. 6
MATERIALS, REAGENTS AND EQUIPMENT NOT SUPPLIED ................................................. 6
SAMPLE REQUIREMENTS ................................................................................................................ 7
WARNINGS AND SAFETY PRECAUTIONS ................................................................................... 7
PROCEDURE ........................................................................................................................................... 8
1.
2.
3.
4.
5.
6.
7.
PCR ............................................................................................................................................ 8
AGAROSE GEL ELECTROPHORESIS .............................................................................................. 9
PURIFICATION OF POSITIVE DRB3/DRB4/DRB5 PCR PRODUCT ............................................... 9
SEQUENCING REACTION ........................................................................................................... 10
PURIFICATION OF SEQUENCING REACTION PRODUCTS.............................................................. 11
DENATURATION & ELECTROPHORESIS OF SEQUENCING REACTION PRODUCTS ........................ 11
EDITING AND ANALYSIS OF ELECTROPHEROGRAMS .................................................................. 12
LIMITATIONS AND CAUTIONS ..................................................................................................... 12
LICENSE .............................................................................................................................................. 13
TROUBLESHOOTING ....................................................................................................................... 13
SUPPORT AND CONTACT DETAILS............................................................................................. 15
Page 2 of 15
For Research Use Only
Principle
The HLA Sequence Based Typing (SBT) procedure described here involves the locus
specific PCR amplification of the HLA-DRB3/DRB4/DRB5 target amplicons which
are visualised by agarose gel electrophoresis. Samples that are positive for HLADRB3/DRB4/DRB5 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 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™
sequence analysis software from Conexio Genomics Pty Ltd.
Page 3 of 15
For Research Use Only
Kit Composition
Kit
PRE-PCR Contents†
(No of vials)
Catalogue No
POST-PCR Contents
(No of vials)
Class II
HLA-DRB3
AN-PD11.0(20)
AN-PD11.0(50)
HLA-DRB4
AN-PD12.0(20)
AN-PD12.0(50)
20 tests
50 tests
20 tests
50 tests
DNA POL – DRB3
1 x 9L
HLA-DRB3 MIX
1 x 370L
DNA POL – DRB3
1 x 18L
HLA-DRB3 MIX
1 x 920L
DNA POL – DRB4
1 x 9L
HLA-DRB4 MIX
1 x 370L
DNA POL – DRB4
1 x 18L
HLA-DRB4 MIX
1 x 920L
Page 4 of 15
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
For Research Use Only
HLA-DRB5
AN-PD13.0(20)
AN-PD13.0(50)
20 tests
50 tests
DNA POL – DRB5
1 x 9L
HLA-DRB5 MIX
1 x 370L
DNA POL – DRB5
1 x 18L
HLA-DRB5 MIX
1 x 920L
†
DRB5EX2F
The PRE-PCR component of each kit consists of a vial/s of a locus-specific PCR mix (e.g.
dNTPs, MgCl2, locus specific PCR primers, and a single vial of DNA polymerase (e.g.
The POST-PCR kit contains sequencing primers (e.g.
DRB3EX2F
DRB5EX2R
1 x 44uL each
DRB5EX3F
DRB5EX2F
DPB5EX2R
1 x 110uL each
DRB5EX3F
) consisting of PCR buffer,
HLA-DRB3 MIX
DNA POL – DRB3
).
).
Page 5 of 15
For Research Use Only
Storage Requirements
The PRE- and POST-PCR boxes may be separated and stored in designated PRE- and POSTPCR freezers. When stored at -20C, the kit components can be used until the expiry date
indicated on the outer kit containers.
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
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
PCR Purification
14.
ExoSAP-IT® (USB® Products Cat No 78200 for 100 reactions)
15.
2mM MgCl2
16.
Shaker
Sequencing, Purification and Denaturation.
17.
BigDye® Terminator Cycle Sequencing Kit v3.1 or v1.1, Applied Biosystems™ by
Life Technologies™.
18.
BigDye® Terminator v1.1 & v3.1 5X Sequencing Buffer, Applied Biosystems™ by
Life Technologies™.
19.
125mM EDTA, pH8.0
Page 6 of 15
For Research Use Only
20.
Absolute Ethanol. Each run needs freshly prepared 80% ethanol solution consisting of
absolute ethanol and sterile water. DO NOT USE DENATURED ETHANOL.
Other PCR and Sequencing purification procedures require validation by the user prior to use.
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 sequencing platforms requires validation by the user prior to use.
HLA Sequencing Analysis Software (eg Assign SBT™, version 3.5 or higher, or
Assign ATF™ (Conexio Genomics Pty Ltd).
23.
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.
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” (eg 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.
Page 7 of 15
For Research Use Only

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.
Procedure
1. PCR
1.1. Set up one reaction for each sample, for each loci 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. 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.
Page 8 of 15
For Research Use Only
2. Agarose Gel Electrophoresis
2.1. Confirm successful amplification of the internal control amplicon in for all DNA
samples tested, and the DRB3/DRB4/DRB5 target amplicon 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 DRB3/DRB4/DRB5 PCR mixes should amplify the
internal control amplicon regardless of HLA-DRB3/DRB4/DRB5 genotype. HLADRB3/DRB4/DRB5 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
DRB3 target amplicon
≈ 640 bp
DRB4 target amplicon
≈ 460 bp
DRB5 target amplicon
≈ 470 bp
Internal control band
≈ 400 bp
Table 2: Expected product sizes for the DRB3/DRB4/DRB5.
3. Purification of Positive DRB3/DRB4/DRB5 PCR Product
NOTE: Purification systems other than EXOSAP-IT® (eg 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. 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 (eg 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.
Page 9 of 15
For Research Use Only
4. Sequencing Reaction
NOTE: Only DRB3/DRB4/DRB5 positive samples identified by gel electrophoresis should
be sequenced using the following procedure.
4.1. Table 3 lists the sequencing primers that are to be used for each locus.
Locus
Sequencing Primers
DRB3
DRB3EX2F
DRB3EX2R
DRB4
DRB4EX2F
DRB4EX2R
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.
Page 10 of 15
For Research Use Only
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 tubes/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 mins.
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 for
no longer than 24 hours.
6. Denaturation & Electrophoresis of Sequencing Reaction Products
Denaturation of extension products
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. Place the reaction tubes on ice for
at least 3 minutes before being placed on the sequencer. If this is not possible, store
at 4C until the reactions reach room temperature or until required.
NOTE: ENSURE THAT THERE ARE NO AIR BUBBLES IN THE REACTION WELLS.
THESE CAN ENTER AND DAMAGE THE CAPILLARY.
Page 11 of 15
For Research Use Only
6.3. Load the reaction plate onto the automated sequencer and prepare the data collection
file according to the sequencer manufacturer specifications.
Electrophoresis Conditions for ABI 3730 & 3730xl automated sequencers
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. Dye set setting for v1.1 Big Dye® Terminator sequencing chemistry).
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 ATF™ software
developed by Conexio Genomics Pty Ltd. Sequence analysis may also be performed using
Assign SBT™ software. For more details please refer to the Conexio Genomics website
(http://www.conexio-genomics.com).
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.

These Sequence Based Typing 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.

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.
Page 12 of 15
For Research Use Only
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.
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.
Check that the appropriate kit
has been used.
Check the thermal cycle
parameters.
Check the negative control for
evidence of contamination.
Decontaminate work area and
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.
Incorrect band sizes
Incorrect kit used
Incorrect
thermal
program used.
PCR contamination
Page 13 of 15
cycling
For Research Use Only
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 14 of 15
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 (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. Consider adjusting
the parameters if an alternative
thermal cycler is used.
Ensure ExoSAP-IT® treatment
is undertaken according to
kit’s user instructions.
Ensure that the PCR mixture is
mixed
thoroughly
with
ExoSAP-IT®.
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 Research Use Only
Problem
Possible cause(s)
Solution
Contaminated sequencing
primer
Presence of Dye blobs
Check sequence quality of the
other sequencing primers and
other samples using the same
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.
Support and Contact Details
Conexio Genomics Pty Ltd
8/31 Pakenham St
Fremantle 6160
Western Australia
Tel: +61-422-863-227
email: support@conexio-genomics.com
Skype: conexiocgx
Website: http://www.conexio-genomics.com
Page 15 of 15
Or your local distributor.
For Research Use Only
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