Development of a Microwave-based DNA Extraction Method to Increase the Success of Direct and Rapid PCR Technique

The goal of this project was to develop a fast, microwave-based extraction technique that could be employed for direct and rapid DNA analysis. The hypothesis was that the use of a microwave could increase the yield of DNA by opening the cell membrane, rendering the DNA available without the use of any other chemical treatment, and improving results from very low quantity samples. At present rapid human DNA analysis is mainly restricted to genotyping saliva and sometimes blood samples. We hypothesized that microwave processing could expand the types of samples assessable to these procedures and increase sensitivity. There were two different microwave ovens employed: a commercial one and a computer-driven microwave. The samples came from three different body fluids: saliva, semen, and blood. Control samples were prepared using the same samples that were not subjected to microwave extraction. The quality and quantity of DNA profiles obtained from these experiments were assessed using a novel PCR-based analysis, a homemade multiplex consisting of 9 simultaneously amplified mini STR loci. A direct PCR method was developed to analyze samples rapidly with minimal pretreatment and no extraction. Once optimized, this procedure was developed for touch DNA samples and mixtures. The optimized microwave protocols were also assessed using a completely automated commercial rapid DNA instrument. We hypothesized that preprocessing by microwave digestion could widen the application of these commercial systems, which are generally designed to analyze oral swabs. The evaluation of the methodologies was performed using statistical tests (ANOVA or t-test) on allele call rates, peak heights, and allelic concordance for all loci. Overall, the use of a microwave coupled with rapid direct PCR represents a valuable addition to an analyst’s toolkit, increasing the DNA yield as well as the speed of the experiments. Moreover, it is less expensive and less toxic than other pretreatments making it an excellent procedure for onsite analysis. This project was supported by the Irregular Warfare Technical Support Directorate (IWTSD, formerly CTTSO) of the US Department of Defense. The research started in October 2019 and was completed in May 202

The Paradox of Government: Explaining the Life and Death of a State

According to Searle (2010), the existence of a State brings a paradox with it. On one side, since a State is a social object, its existence seems to imply the existence of a collective acceptance towards it; on the other side, the existence of this collective acceptance seems to be granted only by the existence of a State that is capable to exercise violence – if needed – on its citizens by means of the military and the police. This implies a contradiction for, if the existence of a government should in principle rely on the free and voluntary acceptance of a certain social system, at the same time it seems that this acceptance derives only from the exercise of brute force, and thus it is all but voluntarily. I will argue that this paradox can be solved only if we distinguish two different notions of collective acceptance: one that can be individuated at the level of natural facts, the other at the level of social – and, more precisely, institutional – facts

Primary familial brain calcification: update on molecular genetics

Primary familial brain calcification is a neuropsychiatric disorder with calcium deposits in the brain, especially in basal ganglia, cerebellum and subcortical white matter. The disease is characterized by a clinical heterogeneity, with a various combination of symptoms that include movement disorders and psychiatric disturbances; asymptomatic patients have been also reported. To date, three causative genes have been found: SLC20A2, PDGFRB and PDGFB. SLC20A2 gene codes for the 'sodium-dependent phosphate transporter 2' (PiT-2), a cell membrane transporters of inorganic phosphate, involved in Pi uptake by cells and maintenance of Pi body levels. Over 40 pathogenic variants of SLC20A2 have been reported, affecting the regulation of Pi homeostasis. It was hypothesized that SLC20A2 mutations cause brain calcification most likely through haploinsufficiency. PDGFRB encodes for the platelet-derived growth factor receptor-β (PDGFRβ), a cell-surface tyrosine-kinase (RTK) receptor that regulates cell proliferation, migration, survival and differentiation. PDGFB encodes for the 'platelet-derived growth factor beta' (PDGFβ), the ligand of PDGFRβ. The loss of function of PDGFRβ and PDGFβ could lead to the impairment of the pericytes function and blood brain barrier integrity, causing vascular and perivascular calcium accumulation. SLC20A2 accounts for about 40 % of familial form and 14 % of sporadic cases, while PDGFRB and PDGFB mutations are likely rare. However, approximately 50 % of patients are not genetically defined and there should be at least another causative gene

Development of a microwave-based extraction for forensic biological samples

In this study, a quick microwave-based treatment was developed as a front end for DNA analysis of forensic samples. The effect of microwave treatment is to cause cell disruption which can improve the release of DNA during direct PCR as well as with extraction methods. Exposure to microwave preprocessing improved the quality of rapid genotyping, particularly when used with low level samples. Optimal results were obtained when samples were microwaved at 300W for 40 s, resulting in improved allele detection. Overall, the addition of this simple preprocessing step improves sensitivity and allele recovery for low level DNA samples when combined with expedited DNA analysis workflows. Its main advantages include speed, low cost, compatibility with downstream DNA methods and application to a wide variety of samples