HLA typing kits from GenoVision are based upon SSPs (Sequence Specific Primers). In our lab, we are mainly using the DQ-DR SSP combi tray for typing bone marrow transplant patients. One DQ-DR SSP combi tray includes a 32-well tray; each well contains primers which are targeted towards a specific internal control and the gene (loci) of interest, PCR master mix (which includes Taq polymerase), buffer, glycerol, nucleotides and even gel loading dye (cresol red).
We first extract the genomic DNA from blood with the Chemagen Genomic Extraction Kit (extraction is based upon magnetic beads). The concentration of DNA is important. If the concentration exceeds 50 ng/ul, it will increase the risk for non-specific amplification, thereby generating weak extra bands. We quantify our final DNA yields with a spectrophotometer.
After extracting the DNA, we just add 3 ul of master mix into well number 32 which is a negative control; we then make the volume 10 ul by adding 7 ul dH2O. Next, we add 74 ul of DNA to a 0.5 ml tube followed by 111 ul PCR master mix and 185 ul dH2O. After mixing well, we dispense 10 ul of the DNA-PCR master mix into each well starting from 1 to 31 and close the tubes with provided caps. Finally, we cycle the reactions as follows: 94ºC for 2 min, (94ºC for 10 sec then 65ºC for 60 sec) x 10 cycles and then (94ºC for 10 sec, 61ºC for 50 sec, 72ºC for 30 sec) x 20 cycles. The total time for completion of the reaction is approximately 1 hour, 5 minutes. The lids should be tightly closed otherwise some of the reagent may evaporate.
After amplification, we load our samples on a 2% agarose gel with 0.5 X TBE buffer (for better band separation). We run the gel for 15-20 minutes and set the voltage at 110 Volts. The voltage and time play a very important role in the analysis of the results. If we run the gel at a higher voltage or increase the duration of the run, there is the chance that one or more bands may run off the gel: the size of some bands is less than 100 bp. It’s very important to run the ladder with your results for better interpretation.
The final step is the interpretation of the results. This is done with SCORE software and is a little cumbersome. However, they do provide an interpretation table with the kit. This table gives approximate size of each PCR product, including controls. This information is helpful in the interpretation of the results.
I faced some initial problems with this kit, like non-specific amplification and loosing the amplified product if I ran the gel for few minutes too long. One drawback I see to this kit is the small volume of amplified product generated (8-9 ul); this is sufficient for only one gel. If you want to repeat the gel run, you need to set up the whole reaction again. However, I think if they increased the reaction volume, it would also increase the price. My last, but not least suggestion is to run at least two lanes of ladder for better analysis.
Once you get the feel of using this kit and gain confidence in interpretation of the results, you will find it to be the easiest method for performing HLA molecular typing.