Cell-Free DNA in Human Blood Plasma

Abstract

Cell-free DNA (cfDNA) in blood is used as a source of genetic material for noninvasive pre-natal and cancer diagnostic assays in clinical practice. Recently we have started a project for new biomarker discovery with a view to developing new noninvasive diagnostic assays. While reviewing literature, it was found that exosomes may be a rich source of biomarkers, because exosomes play an important role in human health and disease. While characterizing exosomes found in human blood plasma, we observed the presence of cfDNA in plasma exosomes. Plasma was obtained from blood drawn into K 3 EDTA tubes. Exosomes were isolated from cell-free plasma using a commercially available kit. Sizing and enumeration of exosomes were done using electron microscopy and NanoSight particle counter. NanoSight and confocal microscopy was used to demonstrate the association between dsDNA and exosomes. DNA extracted from plasma and exosomes was measured by a fluorometric method and a droplet digital PCR (ddPCR) method. Size of extracellular vesicles isolated from plasma was heterogeneous and showed a mean value of 92.6 nm and a mode 39.7 nm. A large proportion of extracellular vesicles isolated from plasma were identified as exo-somes using a fluorescence probe specific for exosomes and three protein markers, Hsp70, CD9 and CD63, that are commonly used to identify exosome fraction. Fluorescence dye that stain dsDNA showed the association between exosomes and dsDNA. Plasma cfDNA concentration analysis showed more than 93% of amplifiable cfDNA in plasma is located in plasma exosomes. Storage of a blood sample showed significant increases in exosome count and exosome DNA concentration. This study provide evidence that a large proportion of plasma cfDNA is localized in exosomes. Exosome release from cells is a metabolic energy dependent process, thus suggesting active release of cfDNA from cells as a source of cfDNA in plasma. PLOS ONE | https://doi.org/10.1371/journal.pone.

Detection and quantification of mutations in the plasma of patients with colorectal tumors

“…This conclusion is consistent with previous studies of other tumor types (21,22) and has important implications for the detection of such mutant molecules.…”

Analysis of the concentrations and size distributions of cell-free DNA in schizophrenia using fluorescence correlation spectroscopy

“…Fragments of ~180 bp (or multiples of 180 bp) are frequently derived from apoptotic cells, whereas fragments larger than ~10,000 bp are from cells undergoing necrosis 12,18 . The increased levels of cfDNA observed in this study suggest that the levels of fragmented DNA were significantly increased in the plasma of schizophrenia patients compared with in the plasma of healthy controls.…”

“…After that, cfDNA has since been described in various diseases in which cell death is thought to have a pathogenic role, including tumors 11,12 , trauma, stroke, myocardial infarction, and sepsis, as well as in physiological conditions such as pregnancy 13–17 . It has been proven by gel electrophoresis, electron microscope, and paired-end sequencing that the most abundant cfDNA fragments in both patients and healthy people were ~180 bp in length, which is a size characteristic of DNA released from cells undergoing apoptosis 12,18 . Quantification and size distribution analysis of cfDNA in different conditions has established numerous new possibilities for both researches into disease mechanisms and biomarker studies 18 .…”

Detection and isolation of circulating exosomes and microvesicles for cancer monitoring and diagnostics using micro-/nano-based devices

“…37 There are somatic mutations only present in tumor cell DNA, which provide a highly specific biomarker that can be specifically detected using quantitative PCR or next-generation sequencing. It was recently demonstrated by Luis Diaz’s group that these DNA fragments can be used for diagnostics and drug-resistance screening.…”