Characterization of circulating DNA as a biomarker for genetic aberrations in humans
Van der Vaart, Maniesh
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Circulating DNA is fragments of DNA which can be found in the blood of healthy as well as diseased individuals. Higher levels of these nucleic acid molecules can be found in diseased and pregnant individuals in contrast to healthy controls. The origin of circulating DNA has not been elucidated, but release of DNA after apoptosis or necrosis or active release by living cells has been hypothesized. It was concluded in this study that apoptosis or necrosis may only be a minor source of circulating DNA and that release of DNA by living cells might play a major role in the origin, while disturbance of the equilibrium between release by living cells and clearance mechanisms may cause the rise in the levels of circulating DNA observed in different conditions. Before circulating DNA can be analyzed, it has to be isolated from the blood. A number of different pre-analytical conditions can have an impact on the quantity and quality of circulating DNA that can be isolated. Furthermore, the choice of isolation and quantification method may also influence the results obtained. Quantitative analysis of circulating DNA was done by real-time PCR analysis of the &Globin gene and the DNA levels obtained for healthy controls and cancer patients correlated with levels reported in the literature. Characterization of total circulating DNA may be beneficial in diagnosis and prognosis and may also contribute to determining the source and function of circulating DNA. In order for characterization to take place a method to clone total circulating DNA was developed and standardized and thirty-five clones were obtained and analyzed. It was found that the sequences contain a large amount of Alu repeats and the significance of this has not been determined yet. This is a first step towards future studies.