A research team, which included scientists from The University of Texas at Dallas and Stanford University School of Medicine, characterized all of the circular DNA in C. elegans, as well as in three human cell types. The study, published online recently in the journal G3: Genes, Genomes and Genetics, found that different cell varieties harbor different sets of circular DNAs.
"The interesting thing is that different types of cells seem to have different repertoires of these circles, even within the same person," said lead study author Massa Shoura, a Beckman Foundation postdoctoral research fellow at Stanford.
"We think they have different functions, and different mechanisms that generate them, but much more study is needed," said Shoura. "One of the things we're trying to find out is whether there are specific repertoires of circular DNA—a term we coined as the 'circulome' —that are specific to various pathologies, like cancer.
In order to establish circular DNA as a biomarker for disease, the team needed a method for reliably and cleanly separating circular DNA out of a sample, purifying it, so that they knew what they were studying was just the circles, without other genetic materials mixed in.
They chose density gradient centrifugation. Developed 50 years ago, the method separates DNA based on density. According to Shoura, this old and rarely used technique is much more effective than modern analysis methods.
"I've been using this DNA isolation technique since I was a graduate student, and I still think it's the best method for recovering a clean sample of circular DNA," said Stephen Levene, co-author on the paper and a bioengineering professor who also is affiliated with the departments of biological sciences and physics at UT Dallas.
In the process, DNA is mixed with a dense salt solution containing cesium chloride in a small test tube, along with a dye that binds to both linear DNA and circular DNA. The dye binds differently to each DNA type, resulting in the linear DNA being less dense than the circular DNA. When the sample is spun in an ultracentrifuge at high speeds, around 120,000 rpm, the higher density circular DNA concentrates in a band near the bottom of the tube.
The researchers also subjected their samples to additional, more modern purification methods to further ensure a clean sample of circular DNA. "This study clearly shows that circular DNA is part of the genome; it plays a role in normal DNA processing," Shoura said. "The more we study it, we're learning that the human genome is more dynamic than we thought."
To purify a sample of DNA and extract the circular DNA, University of Texas at Dallas researchers used a 50-year-old lab technique. They added DNA to a dense salt solution containing cesium chloride in a small test tube, then spun the sample in an ultracentrifuge at very high speeds. Circular DNA is denser and concentrates in a band near the bottom of the tube. Image courtesy of University of Texas at Dallas