This CAREER award by the Chemical and Biological Separations program supports work by Professor Di Gao at University of Pittsburgh to develop innovative DNA separation and mutation screening technologies that are fundamentally different from electrophoresis-based ones. Separation of DNA and mutation detection will be based on pulling DNA strands off a solid surface upon stretching them under the influence of an electric field. The research will investigate the entropic elasticity of DNA molecules upon electrokinetic streching using a dual polarization interferometry technique. Focus will be placed on the elastic properties of DNA in low force (less than 0.1 pN) regimes, single-stranded DNA (ssDNA) secondary structures formed by folding upon itself, and double-stranded DNA (dsDNA) with base-pair mismatches. The force-extension relationship of DNA molecules obtained from these investigations will be employed to design and develop innovative DNA separation and mutation screening processes based on their viscoelastic properties. Processes that evolve from this development will be applied to break through the current limitations of DNA separation technologies in several applications including (i) separation of chromosome-sized DNA molecules, (ii) screening of gene mutations in large DNA fragments, and (iii) separation of DNA sequencing fragments with long read lengths. The research will demonstrate a novel paradigm of separating biopolymers based on their viscoelastic properties, which may also be applied to RNA and proteins.
The outcome of the project may have practical significance in enabling genetic analysis to become part of a standard physical examination and to realize the promise of personalized medical care tailored to an individual's unique genetic identity. In addition to training graduate and undergraduate students, the education component of this proposal consists of three parts: (i) development of a course on biopolymer physics in separation processes for chemical engineering graduate and senior undergraduate students, (ii) development of an international field study module for undergraduate education focusing on international views of ethical and social issues of genetic research through collaboration with Tsinghua University, China, and (iii) outreach to high school and exposure of underrepresented minority pre-college students to science and innovations through development of high-school course unit and workshop by collaboration with Baldwin High School and Westinghouse High School in Pittsburgh with a nearly 100% African-American student population.
We have developed new DNA separation and mutation screening technologies by first tethering DNA molecules to a solid surface and then stretching the DNA molecules with an electric field. By using these technologies, we have been able to (i) overcome the limitation on the size of the DNA molecules that can be separated by electrophoresis-based technologies so that long DNA molecules (such as human genomic DNA of more than 100,000 base pairs) may be separated conveniently and inexpensively, (ii) rapidly separate short ssDNA molecules at a resolution of up to 10 bases in a microchannel without employing polymer matrix to facilitate the separation and recovery of minute amount of DNA for analytical work; and (iii) rapidly screen DNA mutation by examining the viscoelastic properties of ssDNA tethered to a mechanical resonator after it undergoes intra-base pairing and forms a specific complex 3D structure in solution. The capability of rapidly and inexpensively separating both short and long DNA molecules without using polymer matrix greatly facilitates DNA-based diagnosis. The outcome of the project may have practical significance in enabling genetic analysis to become part of a standard physical examination and to realize the promise of personalized medical care tailored to an individual’s unique genetic identity.
