Identifikacija polimorfizama kontrolne regije mitohondrijske DNA u populaciji Bosne i Hercegovine i razvoj protokola za njihovu forenzičku primjenu [Identification of the mitochondrial DNA control region polymorphisms in population of Bosnia and Hercegovina and the development of the protocol for their forensic application]

Aim of this study was to establish locations of the SNP hotspots in the HV 1 region, their alternative allele frequencies, as well as creation of a protocol for their typing based on the single base extension (SBE). ----- Materials and Methods: DNA was extracted from buccal swabs taken from 300 unrelated individuals. Extracted DNA was first quantified using RT-PCR, and then amplified at the HV-1 region of the mtDNA with adequate primers. Resulting amplicons were sequenced by Sanger sequencing using an automated DNA sequencer. Relying on the data thus acquired, primers specific for the chosen hotspots were designed, and samples were typed using SBE method for each SNP in single reactions. After that, designed primers were mixed in multiplex reaction mixes, and samples were typed with those. Created mixed samples from two different DNAs were tested with both singleplex and multiplex reactions. ----- Results: SNP hotspots and their alternative allele frequencies were determined. Taking these in consideration 33 mtDNA types were observed, with Upper Confidence Interval values and Likelihood Ratios calculated for each type. Results obtained from SBE typing protocol of the samples were identical to Sanger sequencing, both in singleplex and multiplex reactions. SBE protocol successfully typed mixed DNA samples. ----- Conclusion: Taking into consideration both the loci and the alternative allele frequencies of selected 15 SNPs, it can be concluded that they make a unique population study, which is shown with comparing calculations of the given mtDNA profile uniqueness using this study data and the data from studies found in the literature. Chosen SNP typing by the SBE protocol has shown itself functional, with results absolutely matching those acquired with Sanger sequencing. Furthermore, SBE protocol has shown high sensitivity in typing mixed DNA samples