Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

Study of circular DNA comes full circle with use of old technique

12.09.2017

A 50-year-old lab technique is helping researchers better understand circular DNA, a lesser-known and poorly understood cousin of the linear version commonly associated with life's genetic blueprint.

With the aid of a process called density gradient centrifugation, a research team, which included scientists from The University of Texas at Dallas and Stanford University School of Medicine, recently published a study that for the first time characterizes all of the circular DNA in the worm C. elegans, as well as in three human cell types.


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.

Credit: University of Texas at Dallas

What Is Circular DNA?

The DNA molecules that make up the genes and chromosomes in our cells are rope-like strands, free at both ends and shaped like a twisted ladder, or helix. That DNA, called chromosomal DNA, is found in each cell's nucleus and contains genetic instructions needed to carry out biological functions.

But another population of DNA, called extrachromosomal circular DNA, is shaped like a circle, with no loose ends, and exists independently of linear DNA. While researchers are beginning to better understand how circular DNA functions in humans, much is still unknown. 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 Dr. Massa Shoura, a Beckman Foundationpostdoctoral research fellow at Stanford who earned two PhDs from UT Dallas. "They're not all created equal -- the circles in your skin cells might be different from those in my skin."

'Circulome' Could Predict Disease

In both the worm and the human cells, the researchers observed circles that were copies of coding regions on chromosomal DNA, regions that contain genes for making specific proteins. While the investigators have some clues as to how such circular DNA is created, the exact processes are not well understood.

"We think they have different functions, and different mechanisms that generate them, but much more study is needed," said Shoura BS'08, MA'10, PhD'13, PhD'14. "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."

For example, Shoura and her colleagues are investigating whether there are marked differences between the circulome in healthy tissue and colon cancer tissue from the same person. If so, circular DNA offers a potential diagnostic biomarker for cancer.

"In order to establish circular DNA as a biomarker for disease, we first have to have a method for reliably and cleanly separating circular DNA out of a sample, purifying it, so that we know what we are studying is just the circles, without other genetic materials mixed in," she said. "That's where my UT Dallas training comes in."

What's Old is New Again

Before she joined the Stanford lab of Nobel laureate Dr. Andrew Fire in 2015, Shoura was a graduate student working at UT Dallas in the lab of Dr. Stephen Levene, Cecil H. and Ida Green Professor in Systems Biology Science. For the study published in G3, which Fire and Levene also co-authored, the team incorporated Levene and Shoura's expertise with an old-school lab technique called density gradient centrifugation. Developed 50 years ago -- and now, according to Levene, rarely used -- the method separates DNA based on density.

Levene and Shoura both said that the technique runs circles around more 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 Levene, a bioengineering professor who also is affiliated with the departments of biological sciencesand 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."

The work was funded primarily by the National Institutes of Health and the National Science Foundation.

UTD Prepared Scientist for Career

Dr. Massa Shoura credits UT Dallas for providing the tools and experience to further her research career. She earned bachelor's and master's degrees in biological sciences from the School of Natural Sciences and Mathematics, and completed doctorates in molecular and cell biology, and in bioengineering, both under Levene's guidance.

"If I was a graduate student now somewhere besides UT Dallas, I might not know what cesium chloride gradients are, and I wouldn't have used them to benefit my research," Shoura said. "There are still not many labs that use this technique, and the ones that do haven't optimized it to work with extremely small samples, as Dr. Levene has done.

"What I accomplished in the Levene lab was a lot of interdisciplinary work in chemistry, math, physics and computer simulation, all applied to DNA," Shoura said. "I'm not an expert in these fields, but by being a little familiar with each one, I am better able to identify a tool or a collaborator that could help on a new problem."

Media Contact

Amanda Siegfried
amanda.siegfried@utdallas.edu
972-883-2155

 @ut_dallas

http://www.utdallas.edu 

Amanda Siegfried | EurekAlert!

Further reports about: DNA cesium chloride chromosomal DNA genes molecular and cell biology technique

More articles from Life Sciences:

nachricht World’s Largest Study on Allergic Rhinitis Reveals new Risk Genes
17.07.2018 | Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt

nachricht Plant mothers talk to their embryos via the hormone auxin
17.07.2018 | Institute of Science and Technology Austria

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: First evidence on the source of extragalactic particles

For the first time ever, scientists have determined the cosmic origin of highest-energy neutrinos. A research group led by IceCube scientist Elisa Resconi, spokesperson of the Collaborative Research Center SFB1258 at the Technical University of Munich (TUM), provides an important piece of evidence that the particles detected by the IceCube neutrino telescope at the South Pole originate from a galaxy four billion light-years away from Earth.

To rule out other origins with certainty, the team led by neutrino physicist Elisa Resconi from the Technical University of Munich and multi-wavelength...

Im Focus: Magnetic vortices: Two independent magnetic skyrmion phases discovered in a single material

For the first time a team of researchers have discovered two different phases of magnetic skyrmions in a single material. Physicists of the Technical Universities of Munich and Dresden and the University of Cologne can now better study and understand the properties of these magnetic structures, which are important for both basic research and applications.

Whirlpools are an everyday experience in a bath tub: When the water is drained a circular vortex is formed. Typically, such whirls are rather stable. Similar...

Im Focus: Breaking the bond: To take part or not?

Physicists working with Roland Wester at the University of Innsbruck have investigated if and how chemical reactions can be influenced by targeted vibrational excitation of the reactants. They were able to demonstrate that excitation with a laser beam does not affect the efficiency of a chemical exchange reaction and that the excited molecular group acts only as a spectator in the reaction.

A frequently used reaction in organic chemistry is nucleophilic substitution. It plays, for example, an important role in in the synthesis of new chemical...

Im Focus: New 2D Spectroscopy Methods

Optical spectroscopy allows investigating the energy structure and dynamic properties of complex quantum systems. Researchers from the University of Würzburg present two new approaches of coherent two-dimensional spectroscopy.

"Put an excitation into the system and observe how it evolves." According to physicist Professor Tobias Brixner, this is the credo of optical spectroscopy....

Im Focus: Chemical reactions in the light of ultrashort X-ray pulses from free-electron lasers

Ultra-short, high-intensity X-ray flashes open the door to the foundations of chemical reactions. Free-electron lasers generate these kinds of pulses, but there is a catch: the pulses vary in duration and energy. An international research team has now presented a solution: Using a ring of 16 detectors and a circularly polarized laser beam, they can determine both factors with attosecond accuracy.

Free-electron lasers (FELs) generate extremely short and intense X-ray flashes. Researchers can use these flashes to resolve structures with diameters on the...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

VideoLinks
Industry & Economy
Event News

Leading experts in Diabetes, Metabolism and Biomedical Engineering discuss Precision Medicine

13.07.2018 | Event News

Conference on Laser Polishing – LaP: Fine Tuning for Surfaces

12.07.2018 | Event News

11th European Wood-based Panel Symposium 2018: Meeting point for the wood-based materials industry

03.07.2018 | Event News

 
Latest News

Microscopic trampoline may help create networks of quantum computers

17.07.2018 | Information Technology

In borophene, boundaries are no barrier

17.07.2018 | Materials Sciences

The role of Sodium for the Enhancement of Solar Cells

17.07.2018 | Power and Electrical Engineering

VideoLinks
Science & Research
Overview of more VideoLinks >>>