SINGLE CELL DNA FRAGMENT SIZING - DESCRIPTION (provided by applicant): We propose to develop a microscope-based imaging system for analysis of bacterial DNA fragments from single bacterial cells. Our approach eliminates the need for cell culturing common to other DNA fingerprinting methods, thereby reducing the analysis time from several days to hours. The proposed technique will allow DNA fragments, from a few hundred base pairs to millions of base pairs, originating from a single cell RFLP, to be sized. We envision many applications of this new capability in biomedicine to: more rapid diagnosis of infectious disease; determination of the source of an infectious disease outbreak; and measurement of the genotoxicity of drugs or environmental agents. In addition, this technique will impact biological research by providing a new measurement tool for single cell DNA analysis, as well as having immediate application to anti-bioterrorism, forensics, food safety, and agriculture. This technology will give researchers a powerful method for studying individual cells and organisms in the absence of averaging effects of ensemble measurements. Likewise, by making measurements on a number of single cells, information about the presence or extent of DNA heterogeneity will be established. The technique relies on performing all sample preparation reactions and analyses in an ultra-thin gel mounted on a microscope slide. Cell lysis, protein digestion, DNA restriction, and DNA staining, along with other reactions, will be carded out by diffusion of reagents into the gel. Staining conditions will be such that the fluorescence intensity is proportional to the fragment size. An electric field will be applied to the gel to electrophoretically separate the DNA fragments. Fluorescence from individual stained and separated fragments will then be detected and quantitated with a microscope-based, high sensitivity imaging system. The resulting DNA fragment size distribution histogram can be used as a fingerprint to identify individual organisms to the level of species and strain, detect damage in the DNA resulting from exposure to ionizing radiation or chemicals, or to monitor genetic variability. To demonstrate this technology we must complete the following specific aims: 1) assemble and characterize the apparatus and measurement approach; 2) develop and optimize the sample preparation chemistry; 3) demonstrate applicability to species and strain identification of representative bacteria.
