Our DNA is under constant attack from many sources: Radiation, ultraviolet light, and contaminants in our food and in our environment can all wreak havoc on our genetic material, potentially leading to cancer and other diseases. Analyzing DNA damage is critical to understanding those diseases, as well as seeking new treatments, but current tools for detecting DNA damage make for tedious and time-consuming work.
Now a team of MIT bioengineers has devised a new way to rapidly reveal DNA damage under a variety of conditions, promising to make such analysis a routine aspect of applications such as drug screening and epidemiological studies of the effects of environmental agents.
The new technique is based on a 30-year-old test known as the “comet assay” — named for the comet-shaped smear that the damaged DNA forms during the test. However, the new technology can analyze a much greater number of cells, at a much faster rate, than the traditional comet assay.
The technology, the result of a collaboration between researchers in the Harvard-MIT Division of Health Sciences and Technology (HST) and MIT’s Department of Biological Engineering, could offer a new approach for epidemiologists to detect dangerous environmental exposures long before they cause cancer, for clinicians to provide better cancer treatment and for researchers in the pharmaceutical industry to identify new drugs and screen out hazardous drugs.
“We expect this could enable studies on a scale that hasn’t been possible before,” says David Wood, a postdoctoral fellow in HST who is lead author of a paper being published in this week’s Proceedings of the National Academy of Sciences that describes the new technique. Wood worked closely on the project with David Weingeist, graduate student in the Department of Biological Engineering.
“A critical feature of our technology is that it can be used to detect genotoxic [mutation-causing] agents in the environment, even if only very basic research equipment is available,” says Bevin Engelward, associate professor of biological engineering and co-senior author of the paper with Sangeeta Bhatia, professor of HST and electrical engineering and computer science and member of the David H. Koch Institute for Integrative Cancer Research and Howard Hughes Medical Institute. “Enabling the prevention of genotoxic exposures in developing nations could ultimately improve the health of millions,” she added.
Chasing comets
The comet assay is based on gel electrophoresis, a commonly used lab test in which an electric field is applied to DNA placed on a slab of polymer gel, forcing the DNA to move across the gel. During electrophoresis, damaged DNA travels farther than undamaged DNA, producing a comet-shaped smear. (The damaged DNA forms the “tail” of the comet.)
This test is generally considered very sensitive, but is also quite laborious. Each experimental condition (for example, a test of how a particular drug affects DNA) requires its own microscope slide, and each slide (containing hundreds of cells) must be visually inspected by the researcher. This limits the number of experimental conditions that can be studied.
To create the new test platform, the researchers from the Bhatia and Engelward laboratories developed the concept of microwells printed into the electrophoresis gel. Using a micropatterning technique developed by Wood and Bhatia, a grid of thousands of tiny indentations is created on the gel. Each cell is placed in its own indentation, giving it an “address” that stays constant throughout the process. Furthermore, each gel can be subdivided into distinct environments by placing a 96-well plate over the gel. Each well confines about a thousand cells, allowing the researchers to add different drugs or other chemicals to each well to compare their effects on DNA damage and repair.
This setup allows dozens of experimental conditions to be tested on just one slide, and it enables slides to be automatically analyzed using custom-designed imaging software.
The new technique is “a major achievement in the development of the comet assay, more than 30 years after its creation,” says Jean Cadet, a DNA researcher at the Center for Atomic Energy in Grenoble, France, who was not involved in the research. Cadet says he anticipates that the new test will become widespread, particularly for epidemiological studies.
The technology was designed to be compatible with basic laboratory equipment, so virtually any laboratory can use it.
To demonstrate the potential usefulness of the new system, the researchers evaluated three compounds that have been proposed as potential inhibitors of DNA repair. Such compounds could be used to boost the effectiveness of chemotherapy agents by preventing cancer cells from repairing the DNA damage caused by chemotherapy. Their study supported earlier predictions that two of the compounds, known as myricetin and DOPA, appear to halt DNA repair, while a third, called NCA, has little effect.
The MIT team is now working with researchers in Thailand to study the effects of air pollutants on DNA in the cells of people who live in highly polluted regions, compared to inhabitants of cleaner areas. In a related study, they are teaming up with researchers at Boston University Medical School to compare cells from smokers and nonsmokers. They also hope to correlate the DNA-repair abilities of individual cells with the genes expressed in those cells.
This work was supported by the MIT Center for Environmental Health Sciences, the National Institute of Environmental Health Sciences (Program in Gene-Environment Interactions), and the National Institutes of Health.
Now a team of MIT bioengineers has devised a new way to rapidly reveal DNA damage under a variety of conditions, promising to make such analysis a routine aspect of applications such as drug screening and epidemiological studies of the effects of environmental agents.
The new technique is based on a 30-year-old test known as the “comet assay” — named for the comet-shaped smear that the damaged DNA forms during the test. However, the new technology can analyze a much greater number of cells, at a much faster rate, than the traditional comet assay.
The technology, the result of a collaboration between researchers in the Harvard-MIT Division of Health Sciences and Technology (HST) and MIT’s Department of Biological Engineering, could offer a new approach for epidemiologists to detect dangerous environmental exposures long before they cause cancer, for clinicians to provide better cancer treatment and for researchers in the pharmaceutical industry to identify new drugs and screen out hazardous drugs.
“We expect this could enable studies on a scale that hasn’t been possible before,” says David Wood, a postdoctoral fellow in HST who is lead author of a paper being published in this week’s Proceedings of the National Academy of Sciences that describes the new technique. Wood worked closely on the project with David Weingeist, graduate student in the Department of Biological Engineering.
“A critical feature of our technology is that it can be used to detect genotoxic [mutation-causing] agents in the environment, even if only very basic research equipment is available,” says Bevin Engelward, associate professor of biological engineering and co-senior author of the paper with Sangeeta Bhatia, professor of HST and electrical engineering and computer science and member of the David H. Koch Institute for Integrative Cancer Research and Howard Hughes Medical Institute. “Enabling the prevention of genotoxic exposures in developing nations could ultimately improve the health of millions,” she added.
Chasing comets
The comet assay is based on gel electrophoresis, a commonly used lab test in which an electric field is applied to DNA placed on a slab of polymer gel, forcing the DNA to move across the gel. During electrophoresis, damaged DNA travels farther than undamaged DNA, producing a comet-shaped smear. (The damaged DNA forms the “tail” of the comet.)
This test is generally considered very sensitive, but is also quite laborious. Each experimental condition (for example, a test of how a particular drug affects DNA) requires its own microscope slide, and each slide (containing hundreds of cells) must be visually inspected by the researcher. This limits the number of experimental conditions that can be studied.
To create the new test platform, the researchers from the Bhatia and Engelward laboratories developed the concept of microwells printed into the electrophoresis gel. Using a micropatterning technique developed by Wood and Bhatia, a grid of thousands of tiny indentations is created on the gel. Each cell is placed in its own indentation, giving it an “address” that stays constant throughout the process. Furthermore, each gel can be subdivided into distinct environments by placing a 96-well plate over the gel. Each well confines about a thousand cells, allowing the researchers to add different drugs or other chemicals to each well to compare their effects on DNA damage and repair.
This setup allows dozens of experimental conditions to be tested on just one slide, and it enables slides to be automatically analyzed using custom-designed imaging software.
The new technique is “a major achievement in the development of the comet assay, more than 30 years after its creation,” says Jean Cadet, a DNA researcher at the Center for Atomic Energy in Grenoble, France, who was not involved in the research. Cadet says he anticipates that the new test will become widespread, particularly for epidemiological studies.
The technology was designed to be compatible with basic laboratory equipment, so virtually any laboratory can use it.
To demonstrate the potential usefulness of the new system, the researchers evaluated three compounds that have been proposed as potential inhibitors of DNA repair. Such compounds could be used to boost the effectiveness of chemotherapy agents by preventing cancer cells from repairing the DNA damage caused by chemotherapy. Their study supported earlier predictions that two of the compounds, known as myricetin and DOPA, appear to halt DNA repair, while a third, called NCA, has little effect.
The MIT team is now working with researchers in Thailand to study the effects of air pollutants on DNA in the cells of people who live in highly polluted regions, compared to inhabitants of cleaner areas. In a related study, they are teaming up with researchers at Boston University Medical School to compare cells from smokers and nonsmokers. They also hope to correlate the DNA-repair abilities of individual cells with the genes expressed in those cells.
This work was supported by the MIT Center for Environmental Health Sciences, the National Institute of Environmental Health Sciences (Program in Gene-Environment Interactions), and the National Institutes of Health.
