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The purpose of this research was to develop high throughput approaches for single molecule and single cell screening for early stage disease diagnosis. High sensitivity as well as high selectivity is essential for the success screening. We first demonstrated a laser-induced fluorescence imaging method that allows screening many single molecules at a time based on their electrophoretic mobilities. YoYo-I labeled DNA molecules were tracked by a charge-coupled device (CCD) camera under a microscope while migrating in the electric field. The purpose is not to separate the DNA molecules but to identify each one on the basis of the measured electrophoretic mobility. Three different procedures were developed to measure the individual molecular mobilities, multi-frame method, streak method, and multi-spot method. Based on their difference in mobility, 2k and 49k bp DNA fragments were unambiguously differentiated. Each measurement only requires a few milliseconds to complete, which opens up the possibility of screening tens of thousands of molecules every second. The results correlate well with normal capillary electrophoresis (CE) experiments for the same samples (2-49 kb dsDNA) under identical separation conditions. The implication is that any electrophoresis protocols from slab gels to CE should be adaptable to single-molecule screening for disease diagnosis. We also developed on-line capillary polymerase chain reaction (PCR) coupled with laser-induced fluorescence detection for individual DNA molecules. A single 30-^m-i.d. fused-silica capillary was used both as the reaction vessel and for isolating single molecules. SYBR green I dye was added into the reaction mixture for dynamic fluorescent labeling. Because of the small inside diameter of the capillary, PCR-amplified DNA fragments from single molecules were localized in the capillary, providing discrete product zones with

High-throughput single molecule screening and selective single molecule PCR for early stage disease diagnosis based on capillary electrophoresis