The PM plus DQA1TM (PE Applied Biosystems) typing kit targets six genetic loci. All six are copied in the initial PCR. The products from this reaction are then placed onto two separate typing strips. One strip is for DQ alpha and the other types the remaining five loci.
There are several steps in a DQ alpha PCR test:
1. DNA from 50 or more cells is extracted. Notice that this test requires fewer cells that the RFLP test. Sensitivity (the number of cells needed) is the main advantage of PCR tests. However, the increased sensitivity also makes PCR tests more vulnerable to trace contaminants, DNA from unexpected sources, in other words.
2. The DNA from the sample is copied over and over resulting in amplification of the original target sequence. The copying or amplification is accomplished in a machine specially designed for this purpose. This machine is called a thermal cycler.
3. The amplified DNA is now treated with a variety of probes that are bound to a blot (see RFLP: Note: In RFLP, the target DNA is bound to the blot and the probe DNA is added. For the DQ alpha dot blot, the probe DNAs are bound to a small blot strip and the target DNA is added).
From the pattern of probes that the amplified DNA binds to, a potential DNA type, also called a genotype, can be inferred.
DQ alpha typing strips look like this before any types are obtained:
The invisible dot to the right of the number 1, has a DNA probe for the 1-allele (variation) for DQ alpha. The invisible dot to the right of the 2 has a DNA probe for the 2-allele and so on. The 1-allele itself has variations, the 1.1,1.2 and 1.3 subtypes, also called alleles. Notice that the typing strip has no specific dot or probe for the 1.2 subtype. Also, the typing strip can't distinguish between the 4.2 and 4.3 subtypes and there is a single dot for these. It is quite possible that there exist DQ alpha alleles that would be undetected by the typing strip and alleles that may be further subtypes of the alleles that the strip does detect.
Here are some examples of how the strips are read:
This last example brings up an important issue with DQ alpha typing. The 1.2 allele is actually the second most common allele in most populations. This means there will be frequent situations where the 1.2 allele may be present but undetected as in the last example. An obvious question is: Why not just have a specific probe for the 1.2 allele? The answer is that the typing strip already maximizes the probing of a relatively short stretch of DNA. That is, the DQ alpha locus itself is only about 240 base pairs long. The multiple probe typing strip was probably about the best that could be done in terms of detecting multiple alleles of this small locus in a single typing step.
Historicall, DQ alpha was often the first PCR-based test that forensic labs used. Actually, the DQ alpha system is quite different from the majority of PCR applications in the scientific community. This will be explained in more detail below.