With regard to the subsequent amplification of the extracted DNA, we read as follows on p.201 of the RTIGF:
“The amplification of the autosomic STRs and of the Y chromosome was performed on the Trace B extract in the manner reported previously on pp. 31, 33 and 34. Trace A however was subjected only to amplification of the autosomic STRs. The results of both amplifications were analyzed via capillary electrophoresis.”
On page 31 of the CT the procedural details of “amplification via PCR” of the autosomic STRs are recorded:
“in order to obtain the DNA profile from the genetic material extracted from the traces [being] analyzed, the following genetic regions (loci) were amplified via the use of pairs of specific oligonucleotide-primers for the regions supporting [fiancheggianti] the various polymorphisms of interest: D3S1358, HumvWA31, D16S539, HumFGA, HumTH01, TPOX, CSF1PO, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, D19S433, D2S1338, amelogenin (sex test) using the “AmpFISTRIdentifier” commercial kit of Applied Biosystem[s] (Foster City, CA) according to the “User Manuals”.
We recall that, with regard to the AmpFISTR Identifiler kit, the instructions provided by the manufacturer for a correct amplification are as follows:
- No. samples x 10.5 μL of AmpFISTR PCR Reaction Mix;
- No. samples x 0.5 μL of AmpliTaq Gold DNA Polymerase;
- No. samples x 5.5 μL of AmpFISTR Identifiler Primer Set;
- 10μL of DNA with concentration ±0.125 ng/μL;
- Final reaction volume 25 μL;
The range of DNA concentration advised by the manufacturer is 0.5-1.25 ng/μL.
In parallel to the samples under examination, controls must be prepared in order to monitor the efficacity of the preselected amplification conditions and/or the presence of contamination.
The controls typically include a “negative control” and a “positive control”.
The “negative control” consists of: PCR amplification Mix (without any template DNA) + water or buffer (used in the preceding phases of extraction and dilution) in the same volumes used for amplification of the traces under examination.
The purpose of inserting the negative control in the amplification phase is to check whether the reagents used for extracting and diluting the DNA obtained from the samples being examined lack DNA — or, in other words, it allows one to verify whether contamination from extraneous DNA is present.
The “positive control” consists of: PCR amplification Mix + standard template DNA with known sequence (provided by the manufacturer of the kit), in the same volumes used for amplification of the traces under examination.
The purpose of the positive control is to make sure that the components and paramenters of the reaction are correct and thus suitable to amplify the regions of DNA of interest; that is, it allows one to monitor the efficacity of the preselected experimental conditions.
Given what is reported in the RTIGF (p. 202) and the absence of specific annotations in the SAL, it must be concluded that no modification was made to the instructions provided by the kit manufacturer regarding the volumes to be used for the amplification reactions; which is to say that a total volume of 25 μL was used, consisting respectively of
15 μL of amplification Mix + 10 μL of extracted DNA
Hence, the amount of DNA used for the PCR reaction was 1.15 ng (0.115 ng/μL x 10 μL = 1.15 ng).
This is an amount which lies within the range suggested by the kits (0.5-1.25 ng/μL of template DNA).