Over the last decade, research in human molecular genetics, fueled by a variety of technological innovations, has undergone enormous advances. Gel electrophoresis, Southern hybridizations, DNA sequencing, and polymerase chain reaction are examples of techniques that have greatly facilitated the discovery and characterization of disease-causing DNA polymorphisms. Unfortunately, the transfer of these technologies from research laboratories into diagnostic clinical laboratories has been difficult, mainly for two reasons. First, many of the abovementioned techniques, especially gel electrophoresis and Southern hybridizations, require skilled personnel and are difficult if not impossible to automate. Second, the technological agendas for research laboratories can be very different from diagnostic laboratories; the former is concerned with discovery and technological flexibility, and the latter with dedicated, automated, high-throughput systems. This project is concerned with a novel gel-free detection system that seeks to unite some of the more cumbersome diagnostic techniques into a single, simple, automated instrument. The technology will allow (1) sizing of small (<200 - 300 bases) DNA fragments, (2) accurate and sensitive probing and detection of amplified or genomic DNA sequences, and (3) sequencing of short stretches of DNA (20 - 50 bases) such as sequence tag sites (STSs). Although such a technology may only have limited application in research laboratories (because of short DNA sizing and sequencing capacities), it is especially attractive to diagnostic laboratories: the sizing and sequencing capacities are sufficient for the detection of polymorphisms, and it is rapid and easily automated for high throughput. Phase I will characterize the parameters and feasibility of the DNA probing, sizing, and sequencing aspects of the technology. In Phase II, automation and scale-up to 96-well format will be proposed. Anticipated Results /Potential Commercial Applications as described by the awardee:The anticipated result of this project is a system that will analyze short polymorphic stretches of DNA by direct sizing, probing, and/or DNA sequencing. The anticipated ease of use, rapid run times, and high throughput of such an instrument should find ready commercial application in clinical diagnostic laboratories involved in the screening of human polymorphisms and genetic diseases. In addition, the instrument's capacity to merge presently disparate genetic diagnostic technologies is expected to accelerate consolidation, standardization, and acceptance of human genetic diagnostic tests.
