Qualitative comparison of DNA isolation methods from insects specimen

Ana Anđelković

Bakalářská práce

Abstract:

Correct identification of the species is one of the most crucial steps in forensic entomology. While morphological identification can be performed, it has a high margin of error, therefore making it a non-reliable source of information in forensic research. Molecular identification based on DNA has proven to be a significantly more accurate method and is the preferred way of identifying insects found on remains. The methods of obtaining the DNA vary in quality of the results, price and ease of handling. In this research, three different methods of extractions were performed and assessed. The DNA was isolated from previously collected and bred specimens of Thanatophilus sinuatus (Fabricius, 1775) and the specimens were stored in 96% EtOH. Two of the assessed methods of extraction were commercially available extraction kits: DNeasy Blood & Tissue Kit (Qiagen), NucleoSpin DNA Insect (Macherey-Nagel), and the third method used was direct PCR. Three different tissue amounts were also assessed throughout all three extraction methods: 1 leg, 2 legs and 3 legs. The amounts isolated were then measured using NanoDrop 1000 UV Visible Spectrophotometer and by Qubit 4 Fluorometer. The extracted DNA was followed by the amplification of the Cytochrome oxidase subunit I (COI) using the forward and reverse primer sequences. Obtained sequences were then visualized using the software Chromas 2.6.6. and blasted against GenBank (http://www.ncbi.nlm.nih.gov/genbank/). The results showed that all three methods had a satisfactory performance in terms of obtaining the desired sequence. The NucleoSpin DNA Insect Kit has shown the best performance in all the assessed parameters, but the price per sample and the requirement of owning not so commonly used laboratory equipment had to be taken into account. The direct PCR method had the lowest success rate, although the price and handling time presented as a significant advantage. This research suggests that, in the field of forensic entomology where precision is of the utmost importance, the NucleoSpin DNA Insect Kit would be the best extraction method, while direct PCR method could be used in situations where the amount of samples is high and the available funds are low.

Keywords

Thanatophilus sinuatus, Forensic entomology, DNA extraction, extraction kits, PCR

Jazyk práce: angličtina

Datum vytvoření / odevzdání či podání práce: 2. 4. 2023

Obhajoba závěrečné práce

• Obhajoba proběhla 23. 5. 2023
• Vedoucí: Ing. Pavel Jakubec, Ph.D.
• Oponent: Jan Růžička, Ph.D.

Česká zemědělská univerzita v Praze

Fakulta životního prostředí

Bakalářský studijní program:
Environmental Engineering