The goal of this research is to develop novel electrophoresis media for large DNA separation (more than 50 kbp) in continuous electric fields, thereby replacing the need to use expensive and slow pulsed-field equipment. The proposed media formulations may obviate the need for pulsed fields by achieving new mechanisms of separation in electrophoresis. The formulations are based on a reversible micro gel technology that allows easy and reproducible formation of matrices. Phase I is directed toward evaluating the feasibility of several proposed formulations for DNA separation. The specific objectives of Phase I are, for each proposed media formulation, (1) to determine synthesis conditions to achieve suitable pore sizes, (2) to determine methods to prepare stable, reproducible matrices, (3) to determine how resolution of large DNA (20 kbp - 2.2 mbp) is affected by pore and particle size in our matrices, and (4) to determine how separations by the matrices compare with current PFE techniques. The most attractive formulation(s) from Phase I will be developed and optimized in Phase II for increased resolution and compatibility with standard techniques of DNA detection and recovery.
