Ricerca di nuovi polimorfismi allelici per l’identificazione personale e caratterizzazione di diverse emoglobine animali ed umane per la discriminazione di specie.

In the first part of my research I investigated new alleles of the human genome. To this purpose I have set up a reliable and repeatable protocol to isolate, amplify and sequence alleles that are employed in Forensic Sciences. The alleles studied in this research, which differ from the most common alleles, are named microvariants. Upon amplification of the microvariant, the two alleles were separates by DHPLC. This approach allows to analyze specifically the selected alleles. In my study I selected nine alleles of the locus D19S433. Four alleles belong to microvariants 6.2. All microvariants showed the same mutation, being a couple of transition the only exception. A deletion of two nucleotides, A and G, at positions 63 and 64 were detected in the sequence G08036, which is composed of 269 pb. These positions are the same detected in most of common alleles, i.e. 10 nucleotides before the repetitive sequence (AAGG)n. Considering the low prevalence in the population of this microvariant at present I cannot consider it as a polymorphism, but rather a mutation that preferentially occurs in the repetitive sequence with the deletion of one or more repetitive units, due to the replicative splippage. Another hypothesis may consider the microvariant 6.2 as a real and new polymorphism, because the sequence of the four microvariants were identical, although rare. In the second part of my research I investigated different globines from animal and human samples. To this purpose I set up and evaluated a protocol of species identification with the separation of the globines from blood haemoglobins. This approach has been published recently for HPLC, while in my study I employed the DHPLC. This analysis allows the creation of a globine pattern for common domestic animals. To reach this goal I analyzed many different aspects, such as sensitivity, reliability and specificity. Data obtained were all positive for the parameters considered. To compare the results I normalized for the EME group the retention time, that should be the same for all the animals. At the end of my research I confirmed the general results with the application of this analysis to two real cases. For both I obtained positive results. Interestingly, in one case, I identified a 60 year old blood trace. These results indicate that my analysis respects the minimal requests to be considered as an alternative method in the Forensic Science Analysis.