Lasota, Tomasz (2014) Purification of human dna for dna profiling from body fluid contaminated soil using superparamagnetic iron oxide nanoparticles (spions). Masters thesis, University of Central Lancashire.
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Extraction of good quality DNA from soil samples is difficult due to the co-extraction of humic acid and phenolic compounds which can inhibit downstream DNA profiling of samples. Several methods are available for DNA extraction from soil including a number of commercially available kits. We used varying amounts (5 mg to 250 mg) of soil contaminated with human body fluid (saliva, semen and blood) to compare commercial kits (Powersoil® and PrepFiler™), a standard phenol/chloroform extraction method, and a method employing superparamagnetic iron oxide nanoparticles (SPIONs) generated in-house. Extraction success and level of purity of the human DNA extracted using these methods were assessed by quantifying the amount of DNA recovered using a Nanodrop 2000 and the amount of humic acid recovered using UV-Vis spectroscopy. Furthermore, successful human DNA profiling was assessed using an in-house generated four-plex PCR assay amplifying fragments of 70 bp, 194 bp, 305 bp and 384 bp, as well as the commercial Powerplex® 16 DNA profiling kit amplifying 16 different amplicons. Our results indicate that the PrepFiler™ kit and phenol/chloroform whilst successful at extracting DNA, contained very high levels of humic acid in the extracted DNA greatly impairing DNA profiling even after dilution of DNA (1 in 10) and addition of higher amounts of Taq (5U). The PowerSoil® kit in contrast, performed best by recovering the most DNA whilst removing the most humic acid, and good quality DNA profiles were obtained without the requirement for dilution of the DNA or addition of extra Taq. However, using extensive optimisation procedures using SPIONs, we have developed a protocol that is able to achieve results very similar to those achieved using Powersoil®, thereby greatly reducing the cost per sample.
|Item Type:||Thesis (Masters)|
|Uncontrolled Keywords (separate with ;):||Forensic science; STR; DNA profiling; DNA extraction from soil; DNA extraction from body fluids; PCR; PCR inhibition; qPCR;|
|Subjects:||C - Biological sciences > C500 - Microbiology|
|Schools:||Faculty of Science and Technology > School of Forensic and Applied Sciences|
|Deposited By:||Paul Harrison|
|Deposited On:||26 Mar 2014 14:29|
|Last Modified:||10 Feb 2017 12:41|
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