Characterization of circulating free DNA in healthy and diseased individuals

Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2009.Circulating DNA is small fragments of DNA present in the blood of vertebrates. Even though the origin, function and significance of these DNA fragments are not elucidated yet, it is widely investigated as a source of biomarkers. for cancer as well as other ailments. This is a result of the detection of a higher concentration of circulating DNA as well as a number of tumor related changes in the blood of cancer patients compared to healthy individuals. The main theories involving the origin of circulating DNA include apoptosis, necrosis and active release of DNA by living cells. A number of arguments against apoptosis and necrosis and in favor of an active release mechanism as the main source of circulating DNA were presented. It was concluded that apoptosis is not the main source of circulating DNA and that an active release mechanism may be a viable alternative. Furthermore, the reason for the increased circulating DNA concentration observed in cancer patients might be the disturbance of the equilibrium between release and clearance of circulating DNA. The utilization and role of circulating DNA as a biomarker for cancer was investigated, and the major methods being utilized to investigate circulating DNA as a biomarker include quantification, detection of genetic alterations and epigenetic analysis of circulating DNA. Comparison of the results obtained by a number of studies lead to the conclusion that circulating DNA is currently overrated as a biomarker for cancer, and before it can be used as an informative biomarker the significance and cause of fluctuation first need to be determined. A method to clone and sequence circulating DNA from cancer patients as well as healthy individuals was utilized in two pilot studies to sequence circulating DNA on a small scale. Analysis of the obtained sequences exemplified the need for large scale sequencing. As a result a method for large scale sequencing of circulating DNA via parallel tagged sequencing on a GS FLX sequencer (454 Life sciences, Roche) were standardized and used to generate sequences obtained from healthy as well as diseased individuals. These sequences were compared between the two groups and to the human genome in an attempt to characterize circulating DNA. The primary aim of this study was the large scale sequencing of circulating DNA, and secondarily, to determine if a significant difference exists between circulating DNA from cancer patients and healthy individuals. A total number of -5500 usable sequences were obtained and analyses of the sequences revealed that, except for a higher frequency of mutations that could be detected in the circulating DNA of cancer patients compared to those from healthy individuals, a significant difference between these two groups were not evident. Thus it can be concluded that this study was successful, all aims were reached, but further sequence analysis will be pursued.Master