Amplification

With regard to the subsequent amplification of the extracts, the following is reported on pages 78-79 of the Technical Report:

“The amplification of the autosomal STRs was performed according to the methods already reported on page 31; the samples which tested positive to quantification (samples A and B) were subjected to amplification and subsequent capillary electrophoresis. The samples which tested negative to quantification (samples C, D, E, F, G) were analyzed following concentration using the Speed-Vac SC110 instrument, ‘Savant’ brand”.

On page 31 of the Technical Report, the “PCR amplification” methodologies are reported:

“with the aim of obtaining a DNA profile from the genetic material extracted from the samples tested, the following genetic regions (loci) were amplified using pairs of oligo-nucleotide primers specific for the regions flanking 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 commercial kit “AmpFISTRIdentifiler” from Applied Biosystem (Foster City, CA) according to the “User Manual”.

It is recalled that the directions for a correct amplification provided by the manufacturer of the AmpFISTR Identifiler kit are the following:

• 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
• 10 μl of DNA with concentration ± 0.125 ng/μl
• Final volume of the reaction 25 μl

The DNA concentration range advised by the manufacturer is 0.5-1.25 ng/μl.

Alongside the samples under examination, controls must be prepared in order to monitor the effectiveness of the selected amplification conditions and/or the existence of contamination.

The controls typically include a “negative control” and a “positive control”.

The “negative control” is constituted 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 to amplify the samples under examination.

The reason for the inclusion of a negative control in the amplification phase is to determine whether the reagents used for the extraction and dilution of the DNA, obtained from the samples under examination, are free of DNA: that is, it allows it to be determined whether contamination from extraneous DNA is present.

The “positive control” is constituted of: “PCR amplification Mix + standard template DNA of known sequence (provided by the manufacturer of the kit), in the same volumes used for the amplification of the samples under examination.

The purpose of the positive control is to ensure that the components and parameters of the reaction are correct, and therefore suitable to amplify the DNA regions of interest: in short, to allow the effectiveness of the selected experimental conditions to be monitored.

Taking into account what was reported in the RTIGF (page 78) and the absence of specific notes in the SAL, it must be considered that no modification was made to the directions provided by the manufacturer of the kit about the volumes to employ for the amplification reaction.

In actuality, from an examination of the documentation in the case file, numerous gaps emerge about the amplification methods.

In particular:

1. the volumes of the Mix are never indicated: it is recognized, in fact, that the equilibrium of a PCR reaction depends on the concentration of the individual reagents used;
2. the quantity of DNA used for each PCR reaction is never indicated: it is equally recognized that best results are obtained when the quantity of template DNA added corresponds to the range indicated by the kit.

Hence the need to preemptively quantify the extract.

Regarding point 2, further clarifications are necessary:

• on page 79 of the RTIGF, it is noted that the samples indicated with the letters C-D-E-F-G (samples, however, which tested negative to DNA quantification) were concentrated using Speed-Vac SC110, but it does not appear that  quantification of the concentrated extracts was repeated. It is noted in the RTIGF that no amplification product was obtained from the aforementioned samples;
• in the absence of any notes about modifications made to the amplification protocols, it must be considered, as noted previously, that the protocols associated with the kit were followed for samples A and B: that is, total volumes of 25 μl were used, constituted respectively of:
• 15 μl of amplification Mix+10 μl of DNA extracted from sample A
• 15 μl of amplification Mix+10 μl of DNA extracted from sample B

With regard to the extract from sample A, if the volumes indicated in the Identifiler Kit were used, it should be assumed that the quantity of DNA used for the PCR reaction was a total of 0.8 ng (0.08 ng/μl  = 0.8 ng): therefore a quantity of DNA which falls within the range suggested by the kit (0.5-1.25 ng/μl of template DNA) and that, as we will see, provided an electrophoretic trace in accordance with the quantity of DNA used for the reaction.

On the other hand, considerable perplexity arises about the quantity of DNA extracted from sample B which would have been added to the reaction mix. The conditional is necessary [1], in that the extract from sample B was quantified using the Qubit Fluorometer™ and although having given an uninterpretable result (“too low”) was considered “positive”, in contrast to sample C which, although being “too low”, was considered negative!

We learn from the transcript of the GUP questioning that Dr. Stefanoni concentrated the volume of the extract from sample B several times.

In particular, she stated having first concentrated the extract from an initial volume of 50 microlitres “to around 20, 22, 23 microlitres” (GUP page 178), and of having subsequently carried out Real Time quantification of the total [amount of] DNA, and not of the DNA of masculine origin.

Since from the Real Time quantification (never carried out!) she obtained a concentration of “a few hundred picograms of DNA” (GUP, page 178) she took steps to further concentrate the extract in order to obtain a final volume of 10 μl which she would have used for the PCR reaction.

We consider that quantification was not even performed on the final volume, since there is no confirmation either in the documentation in the case file (SAL, Real Time report, RTIGF) nor was such a circumstance ever reported by Dr. Stefanoni in the course of her questioning.

In practical terms, an amplification was performed without knowledge of one of the basic parameters: that is, the concentration of the DNA possibly extracted from sample B.

What is surprising is that, given the delicate nature of the testing, quantification of samples B and C was not carried out with Real Time – as happened for samples D-E-F-G – to ascertain if a Low Copy Number (LCN) sample was being dealt with, for which a different and more complex procedure should have been adopted.

The theory that this was a sample which could have been considered LCN was proposed by Dr. Gino in the course of the GUP hearing, and was endorsed by Dr. Stefanoni (“Yes it is possible, certainly!”, page 178) who, although conscious of the problems associated with samples of low DNA quantity (Low Copy Number), did not consider it appropriate to apply any of the precautions recommended by the scientific community in the case of LCN.

It is emphasized that the amplification was performed only once (pages 21-22, transcript of the GUP questioning. To the question, “…the testing of a trace of this sort should be repeated several times to be considered reliable?” The Technical Consultant replies: “In theory yes”. To the question, “How many times did you do it?” she responds, “In this case only once”.

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[1] i.e. the conditional tense, presumably! (“sarebbe stato”)