de Kock, AndréKloppers, Jean F.Soldati, Afika2024-03-142024-03-142023http://hdl.handle.net/11660/12465Dissertation (MMed.Sc. (Haematology and Cell Biology))--University of the Free State, 2023𝗕𝗮𝗰𝗸𝗴𝗿𝗼𝘂𝗻𝗱: Short Tandem Repeat (STR) analysis has proven effective for establishing parentage and biological relatedness. There are commercially available STR kits that allow for reliable PCR amplification and genotyping of STR loci. However, one or two STR loci mismatches may be identified in non-exclusion cases. In routine analysis, these discrepancies are classified as apparent STR loci mismatches. The mismatches result from various mutational mechanisms. However, the mechanisms that drive these mutations are poorly understood. Several STR loci mismatches have previously been reported to impact parentage analysis. The alleles involved in the mismatch affect the interpretation of genetic profiles and can sometimes lead to false parentage exclusions. As such, it is essential to identify and characterise the underlying cause of STR loci mismatches for further validation of the genotypic data produced within a specific DNA profiling laboratory. 𝗠𝗲𝘁𝗵𝗼𝗱𝘀: A laboratory-based descriptive-comparative study was conducted. This study consisted of 100 parentage cases with one or two STR loci mismatches from the DNA testing facility, Universitas Academic Unit, National Health Laboratory Services (NHLS) Bloemfontein from 1 January 2021 to 31 March 2022. The following 15 STR autosomal loci were included in the analysis: CSF1PO, FGA, vWA, D2S1338, D3S1358, D5S818, D6S1043, D7S820, D8S1179, D10S1248, D13S317, D16S539, D18S51, D19S433, and D21S11. Both published and designed study primers were used to optimise the PCR assay conditions for the amplification of the selected STR loci using commercially available control DNA. The optimised PCR assay conditions were used to screen the samples across the 15 STR loci. Sanger sequencing and sequence analysis was conducted for each parentage case to identify and characterise the underlying cause of the observed apparent STR mismatches. Furthermore, the sequence-based alleles were evaluated for concordance with genotypes determined by Capillary Electrophoresis-based (CE-based) STR typing previously reported by the facility. 𝗥𝗲𝘀𝘂𝗹𝘁𝘀: An average concordance of 82% was observed between STR profiling and Sanger sequencing across the 15 STR loci studied. In 11 of the loci, a 100% concordance was obtained. In contrast, no concordance was observed for the D19S433 locus. The stepwise mutations observed at the various loci were 70% more frequent than other mutation models; these were attributed to DNA polymerase slippage. In comparison, 30% of the mutations were as a result of allelic dropouts, accounted for by primer-binding site sequence variants. It was observed that there were more mutations originating from paternal (n=76) rather than maternal (n=26) lineages. 𝗖𝗼𝗻𝗰𝗹𝘂𝘀𝗶𝗼𝗻: The observation of one or two STR loci mismatches in parentage analysis should not be overlooked; all the studied allelic mismatches between the parent and child were characterised successfully. The findings revealed that most of the apparent mismatches occurred due to DNA polymerase slippage. The results of this study provide evidence that sequencing of the core STR repeat and the flanking regions can provide valuable information to characterise STR loci mutational events when inconclusive parentage or kinship results are obtained. Because of the limited sample size, the findings of this study provide evidence that STR mutations are more prevalent in males than females. Furthermore, this study demonstrates the need for DNA testing facilities to have a method in place to characterise and confirm inconclusive genotypic data obtained using the available commercial STR kits.enShort tandem repeats (STRs)non-exclusion casesapparent STR loci mismatchesDNA profilingCapillary Electrophoresis-based (CE-based)DNA polymerase slippageprimer-binding siteDissertationUniversity of the Free State