Development of LAMP-Comet and Immuno-Comet Assays

DESCRIPTION (provided by applicant): The single cell gel electrophoresis (SCGE) or Comet assay, is now internationally recognized as a simple and inexpensive method for the detection of different types of DNA damage, including single- and double- strande d breaks, DNA adducts, cross-links, and alkaline-labile sites in a low throughput format. For the Comet assay, cells are suspended in molten low melting point agarose and layered onto a microscope slide. The cells are then lysed with high salt and detergen t, and the liberated DNA is subjected to alkaline or neutral electrophoresis. Damaged DNA is then detected by its altered migration towards the anode. The individual pattern of DNA migration per cell is called the Comet and is easily visualized by staining with an appropriate fluorescent dye. The Comet assay can be modified for specific applications including detection of acute or chronic genotoxicity, cellular responses to DNA damage, biomonitoring, cancer risk assessment, and cancer cell resistance to tre atment. Two important customized applications for the Comet assay are the detection of cell-marker- and gene- specific DNA damage. The former application utilizes immunofluorescence followed by a Comet assay, and can be invaluable for the screening of drug s that are selectively genotoxic to tumors bearing specific markers, including those produced by tumor viruses. Similarly, assessment of gene-specific damage is useful for the understanding of the effects of replication and transcription on genotoxicity. U sing fluorescence in situ hybridization (FISH), the Comet assay can detect and identified DNA sequences undergoing repair. Currently, the protocols for both of these assays have difficulties associated with them. However, the interest in Comet-FISH has inc reased despite the difficulty associated with this assay. Aim 1 will therefore be to develop a new in situ detection protocol for visualization of newly repaired genes in the Comet assay using loop-mediated amplificatiion (LAMP). Aim 2 will be to develop a new immunostaining method for simultaneous visualization of specific antigens and DNA damage using Quantum Dot Technology. The development of such assays addresses needs to allow rapid analysis of gene and protein-specific DNA damage.