Aim. To detect HLA class I expression variants by SSP in conjunction with the component Null Alleles/Serology project.

Reagents

0.144M ammonium chloride (NH4Cl)

1mM sodium bicarbonate (NaHCO3)

Dissolve 15.4g of NH4Cl and 1.68g of NaHCO3 in two litres of ddH₂0

10mM Tris-HCl pH8.2

0.4M Sodium chloride (NaCl)

2mM di-sodium EDTA pH 8.0 (Na2EDTA)

Dissolve 23.37g of NaCl in 900ml of distilled water. Add 10ml 1M Tris-HCl pH8.2 and 10ml Na2EDTA pH8.0 and make up to one litre with distilled water.

In a fume hood, dissolve 100g of sodium dodecyl sulphate in one litre of ddH₂0. Store at approximately 20^oC to prevent precipitate from forming.

Combine 300ml of NLB with 20ml of 10% w/v SDS. Store at approximately 20ºC to prevent precipitate from forming.

Combine 950ml of absolute ethanol and 50ml ddH₂0.

Combine 700ml of absolute ethanol and 300ml ddH₂0.

Dissolve with warming 350.64g of sodium chloride in 800mls of ddH₂0 and then make up to one litre with ddH₂0. 6M NaCl is a saturated solution so not all of the NaCl will go into solution.

Dissolve 3g of cresol red sodium salt in 500ml ddH₂0. Pass through a 0.22 micron filter and store frozen in 1ml volumes.

Autoclave 2L of ddH₂0. When cold, add 1.5ml/ml each of DRB1 control primers (Olerup 1990) along with 10ml/ml of cresol red and filter through a 0.22 micron filter. The control stock must be tested (see methods) prior to freezing in aliquots.

670mM Tris-base pH8.9

166mM Ammonium sulphate

1%v/v Tween 20 (Polyoxyethylenesorbitan monolaurate)

Dissolve 40.568g Tris base in 400ml dH₂O and adjust to pH 8.9 with conc. HCl. Dissolve 10.96g of ammonium sulphate in the Tris solution. Filtered through a 0.22 micron filter into an autoclaved bottle. Add 5ml of Tween and make up to 500ml with ddH₂0. Store at -70ºC.

33.3ml x10 base buffer

25.1ml 25mM fresh MgCl

140.4ml freshly autoclaved water

1.215ml dNTP mix (i.e. all 4 mixed together)

Test TDMH buffer (see methods) before freezing in 13.3ml aliquots.

To make 2x TBE dissolve 216g of Tris-base and 110g of boric acid in 9L of distilled water. Add 8ml of 0.5M EDTA pH 8.3 and make up to 10L with ddH₂0.

To make 0.5x TBE combine 750ml of distilled water with 250ml of 2xTBE.

10g Electrophoresis grade agarose.

One litre 0.5x TBE

10µl 10mg/ml ethidium bromide.

Dissolve, by heating in a microwave, 10g of agarose in 400ml 0.5xTBE. Top up to one litre with 0.5x TBE and add 10ml of 10mg/ml ethidium bromide solution. Agarose solutions can be stored at 50ºC for up to one week.

The basic tenet of the Phototyping PCR-SSP method is that multiple primer mixes consisting of water, cresol red allele-specific and control-specific primers are synthesised, tested and stored in 1ml primer mix volumes. A typing set collected from these stored primer mixes is dispensed in 3ml volumes under mineral oil in 96-well or 384-well PCR plates. Separate from the primer mixes a PCR buffer (called TDMH) containing all the other ingredients of PCR is made up and stored frozen in aliquots awaiting the addition of DNA and Taq polymerase. DNA is then added to a predetermined volume of the TDMH and 5ml of this mixture is added to each well of the PCR plate prior to PCR amplification and agarose gel electrophoresis. This method allows extreme flexibility in the design and incorporation of any new primer mixes.

One of the key factors in maintaining PCR stringency is the concentration of the primers used: the concentrations given in Table 1 are to be used as a guide only as the optimal concentrations should be determined empirically within individual laboratories, preferably using workshop control DNA samples.

Good quality DNA is paramount for successful PCR-SSP. Sodium citrate or EDTA anticoagulated blood is preferred to heparinised blood as heparin is a severe inhibitor of PCR and especially PCR-SSP (Satsangi et al 1994). If heparinised blood is the only source then the DNA extraction and heparinase protocol described below should allow for satisfactory typing.

This method is modification of Miller's salting-out procedure (Miller et al 1988) where proteinase K is omitted and a chloroform extraction phase is added (Welsh & Bunce 1999). This yields large quantities of good quality DNA in less than 30 minutes which is suitable for PCR-SSP.

To 5ml of anticoagulated blood (not heparinised) add 45ml of RCLB, invert several times and leave to stand for 5 minutes. Centrifuge at 1000g for 10 minutes. Pour off supernatant and gently rinse pellet in 2ml of RCLB and transfer to a 15ml polypropylene tube. The pellet should be white with a pink halo. If there is too much haemoglobin resuspend the pellet in RCLB, agitate and centrifuge. When the pellet is homogeneously white it can be stored at -70ºC or you can continue to the next step. If you are using cell lines pellet the culture in a 15ml polypropylene tube and omit the RCLB stage. Resuspend pellet in 3ml of NLB+SDS (warm NLB+SDS if precipitate visible). Add 1 ml of 6M NaCl, vortex (precipitate should be visible). Add 2ml of chloroform and shake until homogenous milky solution is seen. Centrifuge for 10 minutes at 1000g. Aspirate the DNA (top phase) into a 20ml tube. If the DNA phase is not clear in appearance transfer to a clean polypropylene tube and repeat the chloroform extraction step. Do not suck up any protein from the interface. Add two volumes of 95% ethanol, gently rock until all of the DNA is precipitated. Centrifuge for 5 minutes at 700g and resuspend in 70% ethanol, centrifuge and repeat this washing step. Transfer the DNA precipitate into a sterile 0.5ml microcentrifuge tube, pellet the DNA, and remove the excess ethanol either by centrifugal evaporation, lyophilisation or allowing it to dry on the bench. Resuspend the DNA in 300µl of sterile ddH₂O. From 5ml of blood you can expect to obtain DNA concentrations in the range of 0.2 to 1.0mg/ml. Any DNA sample with a concentration within the 0.2 to 1.0mg/ml range is suitable for PCR-SSP without modification of the DNA volume to be added (see setting up PCR-SSP section).

Tested primer mixes (see notes on batch testing PCR-SSP reagents) should be dispensed in 1ml volumes in 1ml straight tubes which are suitable for placing in standard 96-well format in a 96-well rack. These tubes and racks are suitable for use both with 8/12 channel hand-held electronic multi-dispensing pipettes and also with 96-well robotic dispensers such as the Robbins Hydra. Using a 12 channel electronic dispensing pipette add 10ml of mineral oil to 96 well PCR plates. Dispense 3ml of each primer mix into the appropriate wells of the PCR plates using the Robbins Hydra dispenser. Completed trays may be stored for 6-12 months at -30ºC, preferably in sealed bags or with individual plate sealers.

Thaw out plate(s) containing the primer mixes. Thaw out a 13.3ml aliquot of TDMH and add 64ml of 5units/ml Taq polymerase. This mixture will keep at 4ºC for at least one week. Count how many individual PCR-SSP reactions is required for each individual DNA sample (protocol given here is for 192 reactions). For each 3ml primer mix 5ml of TDMH/DNA/Taq mixture is added. It is important for maintenance of the MgCl concentration that the ratio of TDMH to all other PCR ingredients is 1:0.6. Thus, for 24 reactions add 2.5ml of DNA to 148ml of TDMH/Taq mix. Vortex briefly and pour mixture into a disposable trough. Using an electronic dispenser draw up an appropriate volume. Dispense 5ml of DNA/TDMH/Taq mixture into each well; keep the pipette tip at the top edge of the mineral oil meniscus and allow the mixture to roll of the tip and through the mineral oil. Do not allow the tips to touch the primer mix otherwise carry-over, and consequently false-positive amplifications may occur. On addition of the TDMH mixture to the primer mixes the cresol red will change color from yellow to purple. When the tray is complete, seal with a fresh tray sealer, centrifuge briefly (200g for 5 seconds) to ensure all PCR reactions are mixed and submerged below the oil (vortexing completed plates is not recommended).

Make a 0.2unit/µl solution of heparinase II by adding 50µl of ddH₂O to a 10 unit vial. Add 5µl heparinase per 15µl DNA, agitate and incubate for 90 minutes at 37°C. Add to TDMH mixture as normal. Heparinase activity is destroyed by freeze-thawing.

This program is suitable for the majority of PCR machines:

96ºC for 60 seconds. 5 cycles of 9ºC for 20 secs, 70ºC for 45 secs and 72ºC for 25 secs. 21 cycles of 96ºC for 25 secs, 65ºC for 50 secs, 72ºC for 30 secs. 4 cycles of 96ºC for 30 secs, 55ºC for 60 secs, 72ºC for 90 secs. Cool by ramping to 20ºC for 30 secs prior to termination of the program. Program takes approximately 1.5 hours to run.

Some thermoplastics used for PCR are not an exact fit for every PCR machine and consequently accurate heat transfer to the PCR reaction may be effected. To ensure correct thermodynamics we dip the PCR vessels into a little light paraffin oil, and blot excess on tissues before placing in a PCR machine.

Apply firm and even pressure to the top surface PCR vessels during thermocycling. Preferably use a heated lid.

Use large electrophoresis tanks utilizing gel trays accommodating gel combs with teeth spatially separated for use with multichannel pipettes. Pour 400mls of 1% agarose into the taped off gel tray and insert the combs, allow 20 minutes to set. Fill electrophoresis tank with 2.2L of 0.5x TBE (can be left in tank and re-used at least 15 times). Remove tape and combs and submerge gel tray in tank. Using a multichannel Hamilton syringe add 5ml of orange G loading buffer. Using a multichannel pipette load 18ml of 8 or 12 PCR reactions at a time to the gel (depending on tray layout). Electrophorese for 20 minutes at 200V or until the orange G can be seen to have traveled 3cm.

Visualize the PCR amplicons using 312nm UV transillumination. Record results by gel photography using either Polaroid photography in conjunction with Wratten 22 and 2a filters or any other suitable imaging system. Expose with a shutter speed of 2 and aperture of f5.6. Polaroid type 667 film is suitable for gel photography.

To facilitate identification of positive PCR reactions it is recommended that the electrophoresis lanes are labeled by using an overhead projector (OHP) acetate with the lane numbers printed in the correct spatial orientation. The OHP acetate is laid over the gel in the correct position prior to photography. It is recommended that the OHP is laminated (to prevent wear) and that windows between one row of number and another (where the PCR amplicons appear) are cut out to reduce interference from the plastic fluorescing in UV light.

PCR-SSP interpretation of HLA genotypes is relatively easy, and generally results can be interpreted with little or no prior experience. Each PCR-SSP reaction is deemed to have worked if the control amplification is present. Positive allele-specific amplifications are identified by the presence of a correct sized PCR allele-specific amplicon, whereas absence of an allele-specific amplicon implies absence of the alleles identified in a given primer mix. If a reaction has neither control or allele-specific amplicons the reaction has failed and is deemed "not tested". The alleles that would have been amplified in this reaction are therefore also not tested. Fortunately, many alleles are amplified in more than one reaction so sporadic PCR failures do not often affect full assignation of a genotype. If all of the reactions have failed then the whole result is not tested and must be repeated (see trouble shooting in the notes section). Alleles are assigned by identifying the pattern of positive and negative reactions and interpreting these with reference to the information given in Table 1 (excel file) or PDF file.

The PCR-SSP methods described here are relatively forgiving of pipetting errors however it is wise to frequently check that the pipettes are accurate. PCR machines should be checked periodically for block uniformity by amplifying 96 identical reactions in a 96-well plate and checking for even amplification of both controls and alleles. If you fail to obtain even amplification in all 96 wells the block should be repaired or replaced. Logging which PCR machine is used for individual typing results helps in monitoring for failing machines. Ensure that the PCR plates you are using fit snuggly into the wells of the PCR machine as not all PCR plates and PCR blocks are compatible.

It is essential that all the PCR reagents and consumables are tested for efficiency before routine typing commences. The PCR buffer and its key ingredients such as dNTP's, magnessium chloride and Taq polymerase should be tested for optimal concentration. Some Taq polymerases are more efficient than others so it is important to find an optimal Taq concentration. The ratio of dNTP to magnesium chloride is critical and it is advisable to freeze aliquots of magnesium chloride solution rather than store on the shelf as the magnesium will go off over time.

All primer mixes should be batch tested and stored frozen in suitably sized aliquots. Where possible primer mixes should be tested with both positive and negative samples as well as a no-DNA control for PCR contamination. Periodically all PCR reagents should be tested for contamination: If DNA or PCR amplicon contamination is suspected the reagent must be discarded.

To avoid PCR contamination it is recommended that DNA preparation and pre-PCR steps are performed in a different room to post-PCR manipulation. No laboratory equipment should be moved from the post-PCR room to the pre-PCR room. Obviously gloves used in post-PCR steps should be removed on leaving the post-PCR room. If bench-swab PCR contamination tests are performed in the pre-PCR rooms you must make sure that the swab method has a suitable control for PCR inhibitors.

Getting the right concentration of control primers in the stock control solutions is of vital importance as these solutions are the basis for all the primer mixes. The concentration of primers must be not so high that the allele-specific amplicon is out-competed by the controls. On the other hand, if the concentration of control primers are too low then the control amplicon will be difficult or impossible to visualize and many primer mixes may appear to be not tested. To establish a good working concentration titrate the control primers (suggested titration: 5, 2.5, 1.25ml/ml) in the presence of a constant concentration of a pair of allele-specific primers. Test this titration against some DNA samples of varying quality and of varying genotype (some positive and some negative for the allele-specific primers). The optimal concentration of control primers is found when the control amplicon does not out compete the allele-specific amplicon in HLA allele-positive reactions and yet is present in all allele-negative samples.

The majority of primer mixes will function well using the recommended concentrations given in Table 1 (excel file) or PDF file. However there is some variability in amplification efficiencies between batches of oligonucleotides and primer mixes so that every new synthesis of primer mixes must be properly tested as optimal primer concentrations will fluctuate from batch to batch. If a large volume (20-50ml) of primer mixes are being synthesized for the first time it is advisable to test the recommended concentrations first by making up 0.5ml and testing on appropriate control samples. An ideal primer mix should produce allele-specific amplicons that are easily visible in all expected positive samples and clearly negative in expected negative samples: if possible test some DNA of poor quality as well as normal DNA to ensure a robust primer mix is obtained. If a primer mix is weak or negative with expected positive DNA samples increasing the concentration of primers seldom fails to improve results. Similarly, false-positive amplifications can be removed by titrating either both primers or one of the primers (asymmetric titration).

Each batch of TDMH should be tested in comparison with the previous batch before general use. It is best to test several different DNA samples of different phenotypes to ensure that the buffer is efficient for most primer mixes. The most common defect found when testing TDMH is that the control bands appear strong but the allele bands appear weak, or even non-existent. This is commonly caused by an imbalance in the MgCl:dNTP ratio: either too high a concentration of MgCl or too low a concentration of dNTP's has been used. It is thought that as dNTP's efficiently chelate MgCl an excess of dNTP's sequesters free MgCl and thus deprives Taq of the magnesium that it requires as a co-factor.