Regarding the subsequent amplifcation of the extract for the Y chromosome, on p.34 of the RTIGF we read:
“Analogously to what has already been reported regarding the autosomic STRs (see p.31), in order to obtain the genetic profile (haplotype) of the Y chromosome [of the] DNA specimen of male origin, the following gene regions of interest were amplified via the use of pairs of specific oligonucleotide-primers for the regions supporting [fiancheggianti] the following polymorphisms: DY3S391, DYS439, DYS38911, DYS438, DYS437, DYS19, DYS392, DYS393, DYS390, DYS385, using the “Y-Filer” commerical kit of Applied Biosystems (Foster City, CA) according to the information contained in the relevant “User Manual”. The amplification products were subjected to capillary electrophoresis by means of the ABIPRISM 3130 Genetic Analyzer instrument, using the “Gene Mapper” analysis software.”
We recall that, with respect to the AmpFISTR Yfiler kit, the instructions provided by the manufacturer for a correct interpretation are the following:
- No. samples x 9.2 μL of AmpFISTR Yfiler PCR Reaction Mix.;
- No. samples x 0.8 μL of AmpliTaq Gold DNA Polymerase;
- No. samples x 5 μL of AmFISTR Yfiler Primer Set;
- 10 μL DNA
- Final reaction volume 25 μL;
The range of DNA concentration advised by the manufacturer is 0.5-1.00 ng/μL.
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 volume to be used for the amplification reactions; that is, that a total volume of 25 μL was employed, consisting respectively of:
15 μL of amplification Mix + 10 μL of extracted DNA for the Yfiler.
Therefore, the amount of DNA used for the PCR reaction was 1.15ng (0.115 ng/μL x 10 μL = 1.15 ng).
This amount of DNA lies in the range suggested by the kits (0.5-1.00 ng/μL of template DNA).