Saturday, September 25, 2010

Polymarker (PM)

The PM portion of the PM plus DQA1TM  kit involves 5 genetic loci in addition to DQ alpha. (The manufacturer/ distributor's name has changed from Roche Molecular to PE Applied Biosystems).  These additional loci are named for historical reasons.  The 5 loci are LDLR, GYPA, HBGG, D7S8 and GC.  Each of these represents a distinct location or locus in the DNA.  The 5 non-DQ alpha loci have rather simple allelic variations compared to DQ alpha.  For example, there are only two LDLR alleles detected by the system, allele A and allele B.  The same is true for GYPA and D7S8 that each have A and B alleles that can be detected .  The loci, HBGG and GC each have A, B and C alleles, three variations each in other words.  Thus, reading PM typing strips is fairly simple at least on the surface.  Here are some examples:

 The manufacturer recommends a lower limit of input DNA for PM plus DQA1 typing.  The reason for this lower limit (2 nanograms, ng) is the possibility of missing alleles if the input DNA is too low.  Missing alleles (related terms are "allelic dropout" and differential amplification) is certainly possible in the author's opinion, particularly with low amounts of DNA or degraded DNA.  The phenomenon of failing to detect all alleles present was discussed in the context of DQ alpha in the original User's Guide  although the conditions under which this may occur were not precisely defined with respect to amount of input DNA and the condition of input DNA.  The potential for missing an allele of course increases if the control or S dot is absent or extremely weak, remembering that the C and S dots test whether a threshold has been reached at the PCR and later stages.  The following example illustrates how a DNA profile of one person might change to that of another due to failure to detect an allele. 

 Failure to detect alleles under certain circumstances is a theoretical probability and was actually demonstrated for DQ alpha in the original User's Guide.  The theory that addresses this is called, the "stochastic effect."  In addition to the stochastic effect, a PCR phenomenon called "differential amplification" may play a role when input DNA amounts are low, when input DNA is extensively degraded and possibly at other times. 
 PM plus DQA1 is frequently used on mixed DNA samples from two or more people.  The following example illustrates some of the ambiguity that can arise if interpretations are not cautious:

In the example above, since two of the loci (HBGG and GC) show three alleles, the sample was a mixture of at least two people.  The problem here is that any two people can be included as contributing to the mixture.  The typing strip is saturated, meaning every dot that can be showing is showing.  A poorly recognized limitation of the PM strip is that it is very easily saturated.  For example, two people of types AB/AA/AB/BB/BC (person 1) and AB/BB/AC/AA/AA (person 2) could, when their DNAs are mixed produce the pattern in the example.  In fact, there are almost limitless combinations of 2 types that could produce the pattern.  There are also many combinations of two people that would lead to a typing strip lacking one or two dots.  Finally, there are many mixtures that may mimic a single source of DNA.  For example:

 The profile in this example could have come from a single person whose profile was, AB/AB/AC/AB/AB.  Alternatively, two people of types AA/AB/AC/AA/BB and BB/AB/AA/BB/AA if mixed, could produce the profile.  There are many other possible combinations of people who, when their DNAs are mixed, could produce the profile.  In fact, the only individuals excluded are those possessing the HBGG, B allele and the GC, C allele assuming that the typing strip is reliably detecting all the alleles present.  Extreme caution should be used when there is a possibility of a DNA mixture.  It is arguable whether the system should be relied upon when there is an unresolved mixture.  The ease of saturation may lead to false inclusions.  False exclusions are also possible when the amount of input DNA is low, input DNA is degraded or the S dot is weak or absent.

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