DNA DAMAGE QUANTITATION BY SINGLE MOLECULE LASER SIZING

DESCRIPTION: (Applicant's Description) Using single-molecule sizing by laser-induced fluorescence from DNA moving through a microfabricated channel, this project will develop novel technologies for quantifying DNA damage produced by ionizing radiation and other carcinogens. Damages include DNA strand breaks and oxidative damage to DNA bases, and clusters containing such lesions. This technology will reduce the cost and improve the sensitivity and throughput for quantifying DNA damage. It will quantify low levels of damage and can be applied to DNA damaged in situ, thus facilitating basic research on DNA damage and repair and the relationship of these processes to mutation induction and carcinogenesis. Potential clinical applications include determining the ability of normal and tumor cells to repair damage, thus permitting identification of individuals who may be at elevated risk or the optimum agents to use against a particular cancer. In the R21 phase, we shall assemble a laser system, optics and microfluidic DNA transport system and demonstrate its ability to count single DNA molecules and accurately determine their size. In addition, we shall demonstrate the ability of this system to determine the number average molecular lengths and frequencies of ionizing radiation induced strand breaks in populations of DNA molecules of known lengths. In the R33 phase, the emphasis will shift to focus on the human DNAs from cells and tissues found both in research and clinical settings. We will work with the much larger DNA molecules available from human cells, because the sensitivity of lesion detection increases with the size of the molecules that can be analyzed. We shall also extend the range of lesions that can be quantified, with particular emphasis on double-strand breaks and multiply damaged sites containing heterogeneous mixtures of strand breaks, oxidized bases and adducts, which are difficult for cells to repair accurately. We shall also work towards reducing the quantity of DNA required for a determination of lesion frequency to the level found in individual human cells. The basic technology for single-molecule sizing will be advanced in the R33 phase, first by improving the sensitivity of detection of fluorescence from single DNA molecules by photon counting and phase-sensitive detection. Improvements in fluorescence detection achievable with near-infrared fluorescence will be studied, and near-infrared fluorescent DNA labels developed, leading to a compact system optimized for high sensitivity and throughput and low cost.