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April 24, 2018 - Can these cells repair their DNA? New technology speeds along the answer

Cancer researcher Robert W. Sobol, Ph.D., examines a 96-well CometChip that allows scientists to analyze 42,000 cells at one time for DNA damage and repair.

MOBILE, Alabama (4/24/2018) -- Whether it’s from an occasional after-dinner cigar or a family-friendly day at the beach, the human genome remains under constant attack from outside forces, and that’s on a good day free from environmental pathogens or industrial chemicals.

“Living is a toxic situation,” said Robert W. Sobol, Ph.D., chief of the Molecular and Metabolic Oncology program at USA Mitchell Cancer Institute.

Fortunately, a groundbreaking article co-authored by Sobol and published by Scientific Reports indicates that a recently developed technology provides the tools necessary to determine if a person’s cells – from blood or other organs – are capable of repairing themselves after such damage has been done.

“The cool part about this is it’s not a group assay. We analyze every single cell independently, so the data you get is single-cell analysis, and we’re thinking this is just the beginning because there is so much more we can do to get more information out of those individual cells,” he said.

Sobol, one of eight co-authors on the article “Next generation high throughput DNA damage detection platform for genotoxic compound screening,” said the single-cell analysis technique also allows researchers to determine whether entire groups of cells have sustained damage from invading forces.

“It all comes down to this: We’ve got about 3 billion letters to this ‘instruction booklet’ for the human genome, and they need to be correct. That’s difficult enough under normal circumstances, but then we do these things that are not ideal and cause DNA damage,” he said. “Fortunately for humans – over millions of years of evolution – we’ve got somewhere between 160 and 200 genes whose function it is to recognize damaged DNA and replace it with proper DNA, so the cell can do what it needs to do.”

The sheer volume of analysis required creates a logistical problem, however, and the new technology put forth by Sobol and his colleagues in their paper advances the widely used single-cell gel electrophoresis (SCGE) method, or comet assay. The technology is based on co-investigator Dr. Bevin Engelward’s original 2010 work, in partnership with Drs. Jay George, Sandra Woodgate and Donald G. Lloyd at the biotech firm Trevigen and funded by the National Institute of Environmental Health Sciences.

The new technique creates a 96-well hardware platform coupled with dedicated data-processing software that has proven to increase capacity roughly 200 times over the traditional slide-based SCGE method. The new technique vastly increases “throughput,” or processing rate, for these volume-intensive analyses. The CometChip Platform developed by Trevigen comprises not only the hardware to run the experiment, but also the new software, which is the “brainchild” of Lloyd.

“It is highly reproducible,” Sobol said noting that the new technique holds tremendous promise for human epidemiological studies while also helping to identify and characterize genotoxic agents in large compound libraries.

Specifically, the micro-patterned agarose array employed by this method allows cells to be loaded into individual wells on the slides – enabling the analysis of as many as 400 cells at once – and “achieving a uniform cell distribution in a single focal plane.”

“The software was the real game-changer, because now we can analyze all of that data to quantify all of those comets at once. We’re talking about millions and millions,” Sobol said, noting the roughly 3 million comets analyzed over the course of about three weeks during this study would have taken years using the previously accepted method.

The next step, he said, is to pursue population studies and ultimately offer insight for patients on a case-by-case basis.

“What we want is to be able to apply it to the analysis of regular folks or patients. Do they have elevated DNA damage because of underlying conditions –like diabetes or obesity – or are they being impacted at the genomic level? The day is here when we can now analyze 5 million patient or donor cells in one day and answer some of these questions,” Sobol said.

The first author on the study was Sobol’s MCI colleague Peter Sykora, Ph.D. Co-authors with Sykora and Sobol on the genotoxicity study are Kristine L. Witt, Pooja Revanna, Stephanie L. Smith-Roe, Jonathan Dismukes, Donald G. Lloyd and Bevin P. Engelward. Drs. Witt and Smith-Roe, together with other researchers with the National Toxicology Program, oversaw the creation of the compound plate, and Trevigen provided advanced versions of the 96-Well CometChip® System, the Comet Electrophoresis System (CES), the software -- Comet Analysis System (CAS) -- and the glass-backed CometChips to accomplish the project.


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