Genetic marker typing using PCR

This is the test that was intended to identify which individual the DNA came from. For example, if that human DNA came from a hair falling out of Castanon’s unmasked moustache as he leaned over the sample, we could use this test to determine that the DNA matched Castanon’s and discard the results.

This test does not determine a DNA sequence. The test uses primers to find a specific section of DNA, and amplifies the section between them. The areas selected are STRs, specific parts of the DNA sequence where humans have repetitive sequences, and different people tend to have different numbers of repeats. The amplified DNA can then be sorted by size, so that DNA segments of different lengths form separate bands. Because most individuals will form different combinations of bands, you can compare this pattern with DNA from a known individual to determine who it belongs to.

For example, for the first loci tested, D3S1358, people have between 13 and 20 copies, and you can have a different number on each of your two copies of that chromosome. So you might have 18/19, while I might have 13/17. Which means there are a lot of possible combinations. They’re not all equally likely, so the math gets complicated, but if you test enough loci, and they all match, there are very good odds you’re looking at the same individual. It’s a cheap way to determine if a person’s DNA matches a sample without actually sequencing it (which is expensive).

Because you are looking at repeated regions, it also doesn’t tell you much about the individual. If you got Jesus’s DNA, you would probably want to use it to learn things about him, like his eye or hair colour. However, even for traits where a more in depth DNA test could give you that information, this test can’t, because these regions are mostly non-coding, which means they don’t have an effect on your appearance or health.

The one exception to this is Amelogenin, which can tell you whether it belongs to a male or female. The gene is found on the X and Y chromosomes. The version that occurs on the Y chromosome is slightly shorter, so for people with Y chromosomes you would get two bands, and for people with only X chromosomes you usually only get one band.

The test they used covered the STR loci D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, and Amelogenin, all of which gave no results. Based on that set of loci, I’m assuming they used ThermoFischer’s AmpFlSTR Profiler Plus PCR Amplification Kit, although the report does not specify.

This kit recommends a DNA sample of 1.0-2.5ng, although they say smaller samples have been successfully profiled. Anything below 0.1-0.2ng is considered “low template DNA” and starts to experience stochastic effects, which means you can get different results each time the test is run on the same sample. The report clearly indicates that based on the quantiblot test, the researchers did not think they had enough human DNA to get a profile:

“Insufficient DNA for successful PCR analysis was obtained from item #1-2. As requested, PCR STR analysis was attempted on this sample but no reportable genetic profile was obtained.”

So, contrary to the narrative Tesoriero presents, it’s not surprising that there the quantity of DNA found by the Quantiblot was low enough that the researchers weren’t expecting a result from the PCR STR, and they didn’t get one.

I find Tesoriero’s portrayal of this situation as a mystery outright dishonest. The point of the Quantiblot was to determine if there was enough human DNA for the PCR STR to work. It found that there probably wasn’t, they tried anyway and it failed. That’s the predicted result. I see no indication that anyone was surprised by this other than Tesoriero.

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