DNA barcoding is a broad-spectrum technique applicable in taxonomy, agronomy, evolutionary biology, and other fields. It is based on identifying living and deceased organisms through a standard region characteristic of a particular group of organisms. For most animal species the 5' end of the gene, encoding cytochrome c oxidase subunit 1, was selected as the barcoding region. Slovenia harbors a diverse range of scorpion species. Representatives of genera Euscorpius and Alpiscorpius inhabit the country, including high-altitude regions. According to current taxonomy, there are ten or eleven species present in Slovenia. None of the species found in Slovenia pose a threat to human life. We isolated total DNA from scorpions and used Folmer's universal primers, LCO1490 and HCO2198, to amplify the DNA barcode region. In this thesis, we aimed to optimize the DNA isolation process from ethanol-preserved scorpions to obtain high-quality DNA, enabling a successful amplification of the previously mentioned region. With these amplified fragments, we could directly determine the nucleotide sequence without prior vector ligation and bacterial cloning. We directly sequenced amplicons that were not of scorpion origin, but belonged to the owlet moth genus Chrysodeixis. This was likely a consequence of DNA contamination. Due to difficulties with isolation and direct sequencing, we also performed a comparison of the efficacy of two reagent kits for DNA isolation, an investigation of the source of non-scorpion sequences discovered whilst DNA barcoding, and an evaluation of the PCR amplification efficacy using two pairs of Folmer's universal primers. We have found that age and storage of reagents kits and primers have a significant impact on the success of determining the DNA barcode and that two PCR buffers that are frequently used in our lab are most likely sources of contamination, resulting in non-specific amplification. Furthermore, we presented an alternative to direct nucleotide sequencencing involving cloning, sequencing and determination of the DNA barcode within a selected vector. We determined the nucleotide sequence of inserts and once again confirmed the infection of the samples with insect DNA from owlet moths.
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