The extraction of DNA from the items under examination occurred on the same day of 2/22/2011, in another laboratory assigned to the phase of DNA extraction, physically separated from the areas where forensic samples are prepared and amplified.
The counter, as well as all equipment and laboratory instruments, were first treated with 10% sodium hypochlorite solution, washed with water and finally with 70% ethanol, as unanimously recommended and practiced by molecular biology laboratories.
Prior to autoclave treatment, four sealed packages, each containing two metal tweezers and two clippers [forbicine], were first prepared; as were, in addition, two Becker containers, the first containing a 10% sodium hypochlorite solution for the disinfection of the instruments in use, and the second sterile bidistilled water for the rinsing of the instruments prior to the next use.
All the operations were carried out while wearing masks, disposable gowns, and disposable gloves which were regularly changed between one sample and the next.
On each individual swab (12 swabs in total including the extraction-negative [il negativo di estrazione], labeled with the letters from A to M in the case of the specimens taken from the items, and with the marking “C.N.” for the negative control) we proceeded to take a sample of a piece of cloth [tessuto], of dimensions about 2×2 mm, for the subsequent cytological analysis.
The pieces of swab taken for the cytological analysis were separately inserted into 1.5 mL tubes (each of which labeled on its top with the letter corresponding to the respective sample).
The tubes were placed inside a carton that was closed by means of brown-colored adhesive tape on which the signatures of the experts and the consultants for the parties were placed. The carton was refrigerated at +4°C until the time of the next laboratory tests.
The remaining swabs were removed by means of sterile tweezers and clippers, individually inserted into 1.5 mL tubes (on the top of which was placed the letter corresponding to the sample in question) and then subjected to the DNA extraction procedure by means of the commercial kit labeled DNA IQ™ System (Promega Corporation, Madison, WI, USA), specifically designed for forensic genetics laboratories, which enables DNA to be isolated via paramagnetic particles and can be used for extracting DNA from a large variety of samples, including dried traces and biological liquids.
For the extraction of DNA from the samples, the following protocol, indicated in the user’s manual of the kit, was followed:
1) removal [prelievo] of the swab and insertion into a 1.5 mL tube;
2) addition to each individual tube of of 250 μL of Lysis Buffer (preliminarily prepared via the addition of 1 μL of 1M DTT for each 100 μL of Lysis Buffer) and incubation at 70°C for 30 min.;
3) transfer of the Lysis Buffer and of the sample to a new 1.5 mL tube equipped with a centrifuge basket [cestello per centrifuga] and subsequent centrifugation at maximum velocity (13000 rpm) for 2 min.;
4) removal [rimozione] of the basket; addition of 7 μL of magnetic resin; vortex and incubation at room temperature for 5 min;
5) vortex and positioning of the tube on the magnetic support; discharge [rimozione] of the solution;
6) lavage with 100 μL of Lysis Buffer (working solution [sol. di lavoro]);
7) lavage with 100 μL of Wash Buffer 1X (previously prepared via the addition of 15 mL of absolute ethanol [etanolo assoluto] and 15 mL of isopropanol to the Wash Buffer 2X included in the kit), repeated for a total of 3 lavages.
8 ) accurate removal [rimozione] of the solution; drying with open top, on the magnetic support, at room temperature, for 5-10 min.;
9) addition of 30 μL of Elution Buffer and incubation at 65°C for 5 min.;
10) vortex and repositioning of the tube on the magnetic support;
11) transfer of the solution into a new tube;
12) storage of the extract at +4°C.
We note that the samples under examination were rigorously subjected to the DNA extraction procedures individually according to a process that was precisely organized in terms of location and timing [secondo una precisa organizzazione temporo-spaziale della lavorazione], using disposable sterile filter-equipped tips. The operations were conducted while wearing masks, disposable gowns, and disposable gloves which were regularly changed upon each passage of the process from one sample to the next.
At the end of the extraction procedure, the 12 1.5 mL tubes (each of which labeled on the top with the letter corresponding to the sample in question) were placed inside an appropriately identified carton, sealed with brown-colored adhesive tape on which were placed the signatures of the experts and the consultants for the parties, and refrigerated at +4°C pending the quantification of the extracted DNA.
At 6:05 pm the testing operations were interrupted; they resumed the next day 3/23/2011 at 10:00 am according to the memorandum which we quote below:
“The next day, 03/23/2011, at 10:00 am, Dr. Stefanoni, Dr. Gino, Dr. Onofri, as well as the appointed experts Prof. Vecchiotti and Prof. Conti were present; after checking the integrity of the adhesive tape of the container of the extracts performed on 3/22/2011, all reported to the histology section of the appointed experts’ home Department. In the presence of the parties, preparations were made for a quantification reaction via Realtime 7500 PCR; at the end of the quantification, the results were viewed; they were then printed, and copies were given to the consultants present. Dr. Stefanoni notes that the Real Time PCR reaction was prepared on the counter without using a fume hood to guarantee the absence of contamination. Dr. Gino has nothing to state. Dr. Onofri has nothing to state. This memorandum ends at 2:15 pm.”