Cell-free DNA (cfDNA) is released into the bloodstream by a combination of apoptosis, necrosis, and secretion within membrane-bound vesicles^13,14,15. The genetic and epigenetic modifications of an individual cfDNA molecule reflect the changes present in the cell of origin^16,17,18. In healthy individuals, the majority of cfDNA is released from haematopoietic cells^18,19, though pathological tissues have been observed to increase their relative cfDNA release²⁰. The half-life of circulating DNA in plasma has been measured as between 16 minutes and 2 hours^21–23, due to degradation and renal excretion²⁴.

 

cfDNA molecules released by apoptosis have a modal length of 166bp, corresponding to the length of DNA wrapped around a nucleosome plus its linker^25,26, reviewed by Jiang and Lo¹¹. Evidence suggests that DNA bound to proteins or transcription factors preferentially survives digestion, while naked DNA is degraded¹⁷. Fragment size profiles of cfDNA demonstrate approximate periodicity of 10bp²⁷, caused by nucleases cleaving the DNA strand at periodically exposed sites corresponding to the helical pitch of the double-helix (~10Å, 10bp)²⁸.

 

The detection of cfDNA and ctDNA has been shown in a variety of body fluids other than blood, including urine^24,29, CSF^30,31–33 and pleural fluid³⁴, which may have utility for the study of cancers in organs such as bladder, brain, or head and neck, where these fluids are most proximal and may contain higher levels of DNA of tumour origin, given the evidence for direct shedding of cfDNA³⁵.