Quantification of DNA

Quantification of the DNA extracted from the aforementioned samples was then performed.

On page 78 of the RTIGF, the following tables are shown:

The above tables show that DNA quantification for all samples was performed with Real Time PCR, using the 7700 Sequence Detector ABI PRISM™ apparatus from the company Applied Biosystems.

On the other hand, no information is given about the kit used for the DNA quantification.

Regarding the quantification result for the samples indicated by the letters A and B, the expression “positive” is reported, but no numerical value is given for the DNA detected; conversely, the quantification result was “negative” for the samples indicated with the letters C-D-E-F-G.

It should be emphasized that from an examination of the Real Time PCR reports exhibited, it appears that quantification using this method was only carried out, on 18 December 2007, on the DNA extracts indicated with the following numbers:

48649 = sample D

48651 = sample E

48654 = sample F

48655 = sample G

These samples all tested negative for the presence of DNA (c.f. Real Time Report DNA=0.00).

A report is also attached relating to quantification of the extracts from the [following] samples, dated 13 November 2007:

  1. A (sample code ID 47329)
  2. B (sample code ID 47330)
  3. C (sample code ID 47331)

[This was] performed using the Qubit Fluorometer™ from the company Invitrogen, and using the dsDNA HS Kit. This is a selective test for double-stranded DNA, but not specific for human DNA. Samples with a DNA concentration range between 0.2-100 ng can be accurately and easily quantified.

The attached card shows that the following reagents were prepared:

  • Stock solution: for each sample 199 μl of Buffer and 1 μl of reagent;
  • Standard (1 and 2): 190 μl of stock solution and 10 μl of standard;
  • For each sample: 199 μl of stock solution and 1 μl of sample.

Using the Qubit Fluorometer™, the following concentration values for the samples were obtained:

  • Sample A (sample code ID 47329): 0.08 ng/μl
  • Sample B (sample code ID 47330): too low
  • Sample C (sample code ID 47331): too low

From this, it can be inferred that what is reported on page 78 of the RTIGF (and confirmed in the GUP questioning, page 178, where it is claimed that quantification was performed using Real Time and that quantification of the Y [chromosome] was not carried out) is not consistent with what was performed in reality. In fact, the tables show that quantification of the DNA extracted from all the samples taken from Exhibit 36 was carried out using the 770 Sequence Detector ABI PRISM™ equipment (Applied Biosystems); in contrast, it is apparent from the attached cards that the aforementioned method was only performed on samples D-E-F-G. A different method was employed for samples A-B-C using the Qubit Fluorometer™, and not mentioned in the final Technical Report, as would have been due and correct.

Regarding the interpretation of the quantification,  it should be noted that on page 78, the following is stated: “the samples testing positive to quantification (samples A and B) were subjected to amplification and subsequent capillary electrophoresis…”.

As regards the quantification of sample A (knife handle) the results obtained with the Qubit Fluorimeter™ show that the concentration of DNA in this sample was equal to 0.08 ng/μl. Taking into account that the “quantity of extract” was 50 μl (c.f. SAL), and multiplying 0.08 ng/μl x 50 μl, the total [amount] of DNA was equal to 4 ng: certainly a significant quantity, which allowed sample A to be considered positive to quantification.

On the other hand, it is not possible to comprehend the criteria adopted in the assessment of the positive quantification result for sample B and the negative result for sample C, given that the same result, “too low”, was obtained for both samples: that is, a value which must be considered not only below the sensitivity threshold of the Fluorimeter indicated by the manual (DNA concentrations equal to 0.2 ng/μl), but below 0.08 ng/μl, a value which the Fluorimeter detected for sample A.

Nor is it comprehensible, considering the negative results on sample B, what Dr. Stefanoni reported during the GUP questioning (page 178) where she stated that the DNA in sample B, quantified with Real Time PCR (it is recalled that such quantification as confirmed during the hearing was never carried out or, at least, no documentation was provided to support this claim), was “in the order of some hundreds of picograms”, a value which does not appear in any of the documents provided to us (SAL, Fluorimeter report, Real Time report, RTIGF).

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