Molecular techniques including diagnostics that require separation or identification of different sized nucleic acid sequences have remained unchanged over the last several decades and rely primarily on some form of electrophoresis. Diagnostics for several human diseases, such as those associated with trinucleotide repeat instability, exemplified by Fragile X syndrome, currently require these conventional techniques. These conventional techniques have inherent disadvantages of being labor intensive and less amenable to automation, and also not suitable for being developed into screening platforms. We have been developing a unique technology for DNA separation and fractionation that overcomes many of the limitations of current DNA separation technologies, such as electrophoresis. The technology is based on first tethering the DNA molecule to a solid surface through precise end-hybridization and then sequentially pulling the DNA off the surface under an electric field. The anchor is such designed that the critical force to detach a DNA chain is independent of the chain length. Because the electrical force is proportional to the DNA net charge, a gradual increase of the electric field leads to size-based selection of the DNA strands-longer DNA molecules depart the surface first followed by the shorter ones. Significantly, this highly efficient method does not require use of a matrix (like agarose or PAGE) and has no upper or lower limit on the length of DNA that can be separated. This innovative DNA separation method is now being proposed to be developed into an instrument that can serve as a key / platform technology, primarily for the diagnosis of trinucleotide repeat instability associated genetic diseases diagnostics and screening. The device and technology was developed at the University of Pittsburgh, but has not yet been available to other researchers beyond the original investigators. In this project, a commercial development of this novel idea is planned that it will make a significant impact in a wide variety of clinical and research scenarios.
Molecular techniques including diagnostics that require separation or identification of different sized nucleic acid sequences have remained unchanged over the last several decades and rely primarily on some form of electrophoresis. We propose to develop a novel instrument using electric fields to separate long double-stranded DNA molecules from shorter ones based on immobilization of DNA molecules on a glass slide through short anchors attached to its surface. Significantly, this highly efficient method does not depend on a matrix (like agarose or PAGE) and has no upper or lower limit on the length of DNA that can be separated. This innovative DNA separation method is now being proposed to be developed into an instrument that can serve as a key / platform technology, with a focus on the trinucleotide repeat instability associated genetic diseases diagnostics.
