PROJECT: A New Comet Assay for Measuring DNA Damage by Space Radiation
PI: Cheng Fu Chen, Professor, UAF
PI: Andrej Podlutsky, Associate Professor, UAF
Space radiation detrimentally affects biological systems and individual organisms. Earth’s magnetic field provides protection to terrestrial inhabitants. However, space missions beyond the low Earth orbit poses increased risk to the health of humans because of radiation in space. Both components of space radiation, chronic exposure to low-dose to galactic cosmic rays and random short-term exposures to the energetic particles from the Sun, might increase risk of malignant transformation. Damage to the genetic material of cells, its DNA, is considered to be the first and most important step in the malignant transformation of normal cell into cancerous one. As this concern is addressed in NASA’s 2012 roadmap in the domain of Human Health, Life Support and Habitation Systems (HLHS), however, currently we do not have reliable techniques to measure DNA damage on a board of spacecraft. A reliable and sensitive technique to collect data at ground and in flight for radiation risk assessment is in demand by NASA.
When exposed to radiation, single and double strand-breaks in cellular DNA are produced. The amount of strand breaks can therefore be correlated to the extent of damage to cells under radiation. One of the most convenient models to study DNA damage and repair process in the live cells is the comet assay, or single cell gel electrophoresis technique. This technique is currently configured to conduct a measurement on live cells under laboratory set-ups. Therefore, quite often the existing comet assay is not suitable for “in field” applications. Development of a simple and sensitive technique would allow researchers to study crucial cellular processes outside of laboratory boundaries and open possibilities for evaluating many environmental exposures currently unobtainable, such as DNA damage during short space flight or long-term interplanetary explorations within the scope of NASA’s missions.
In this project we are aimed at developing a new technique, we call it vertical comet or v-comet, to measure the level of DNA damage in live cells or fix-preserved cellular DNA samples. (Here for fix-preserved DNA sample we mean a DNA-agarose mixture obtained per the steps, first embed live cells in agarose and then lysis the cells to form a DNAagarose mixture.) This new assay will be prototyped and tested using the microfluidics technique. The development in microfluidics techniques has demonstrated numerous potentials of microscale analysis for biological studies, and has shown some advantages of reducing labor and risk in contamination. We will (a) develop a microchamber containing live cells or preserved cellular DNA; (b) use electrophoresis to sample damaged DNA by pulling broken strands into a circulating buffer in a closed-loop; (c) electrophoresis is continuously applied while the buffer is circulating to accumulate the broken strands; and (d) the amount of DNA will be measured by a sensitive fluorescent labeling. By taking the advantages of small scales of microchamber configuration, in this proposal we will design and test an integrated approach combining sensitivity of traditional comet-assay technique with the versatility and subtle precision of microfluidics technique.
We envision that this new technique will allow researchers to measure DNA damage in the field conditions. One ready application of the new assay would be to help astronauts avoid overexposure to space radiation. Another apparent application is on the construction of a correlation between the doses of chronic space radiation and the extent of cumulative DNA damage. An additional, significant contribution of the research outcome would be the innovation of a v-comet assay on either live cells or sampled cells in a conserved, dried platform to study DNA damage not bounded by the laboratory environment.