This Small Business Innovation Research (SBIR) Phase I research project will develop a novel DNA-conjugated marker (drag-label) in genetic fragment size analysis using free solution electrophoresis. This method uses no sieving matrix, so the separation occurs in a fundamentally different manner than using an entangled polymer solution as a sieving matrix and offers the advantage of faster separation with shorter separation distances required. The improved drag-labels must be readily conjugated to DNA fragments and have sufficiently low size variance to allow separation of the DNA sequencing fragments by charge rather than size. The research project will test a drag-label after it is conjugated to PCR primer for short tandem repeat (STR) fragment size analysis and dye-labeled for fluorescence detection. The utility of this method using the proposed drag-label will be investigated and electrophoresis separation performance evaluated to determine the parameters limiting separation efficiency. In addition, the ability to discriminate based on relative intensities after varying the number of dye molecules on the drag-labels of different sized fragments will be evaluated. Commercially, the application is improved fragment size analysis using free-solution electrophoresis. This approach offers significant improvement in separation speed and efficiency (cost reduction), with increased sensitivity using multiple dye-conjugates on each drag-label. DNA sequencing using capillary electrophoresis with an entangled polymer solution as the sieving matrix has experienced tremendous advances in the last decade. New technologies for sequencing are now promising even greater sequencing capabilities. However, these new sequencing technologies are not useful for genetic fragment size analysis methods, such as forensics human identity using STR analysis and other applications using RFLP analysis. Improved technologies are needed for these applications. This method for free-solution electrophoresis for forensics applications can be incorporated into existing capillary electrophoresis instrumentation, allowing rapid and low cost implementation
