P39a: The characterization of total circulating DNA from blood of healthy donors and cancer patients

Abstract

Background It was shown that circulating DNA could be used as a valuable source of material for cancer diagnostics (Fleischhacker, 2007), but unknown and unpredictable factors frequently dramatically decrease concentration of tumor-specific genetic biomarkers by influencing on generation and circulation in bloodstream of extracellular DNA. Sequencing of total circulating DNA could either provide valuable information regarding such factors or determine novel targets (DNA markers) for cancer diagnostics. Materials and Methods We used a Sanger’s technology (ABI PRIZM 3110 sequenator) and BLAST analysis of circulating DNA from plasma and cell-surface-bound fraction of healthy individuals and patients with breast cancer. DNA was isolated by guanidine thiocyanate/glass milk method (Tamkovich, 2004) followed by Sau3A and EcoRI hydrolysis. Fragmented DNA was ligated into the pBlueScript II KS(-) vector. Competent XL-Blue ( E. coli ) cells were transformed with the vector by electroporation and after recovery the cells were plated on LB+Amp agar plates. The inserts length estimate by PCR-analysis. Results The majority of investigated circulating DNA fragments were about 100–800 bp. Sequence analysis revealed that number of circulating DNA fragments does not depend from the size of paternal chromosome both in normal and pathological state, but fragments from chromosome six were found in the female bloodstream more frequently. It was found, that in cancer patients blood were observed frequency of occurrence of pseudo genes and CpG islands below then in healthy female blood. BLAST analysis demonstrates that 10% of coding DNA fragments circulating in blood of the breast cancer patients is associated with development of this pathology. Conclusion Sequencing of circulating DNA demonstrates a variable concentration of different DNA sequences in comparison with genomic DNA. Further characterization of circulating DNA may be beneficial in diagnosis and prognosis and may also contribute to determining the source and function of circulating DNA.