Increasing the rate of DNA restriction mapping and sequencing is one of the major obstacles to be overcome in efforts to map the human genome. Capillary electrophoresis provides a means of enhancing the speed of a separation compared to conventional slab gel electrophoresis, but is presently restricted to small DNA fragments. We have developed a capillary electrophoresis technique using uncrosslinked hydroxyethyl cellulose which has shown the ability to separate DNA fragments up to l,400 bp in length with a resolution of 10 bp, and larger DNA fragments up to 23,000 bp with more limited resolution. Based on our present theoretical model of the mechanism of separation, this upper base-pair limit can be considerably extended, and resolution much improved. The first set of objectives of this proposal is thus: (l) To determine the mechanism of DNA separations in dilute, uncrosslinked polymer solutions. (2) To investigate the upper limit of DNA size which can be separated by capillary electrophoresis in uncrosslinked polymer solutions, and to extend the usefulness of CE to separations of DNA > 50 kbp. (3) To correlate sieving polymer properties, such as molecular weight, stiffness, and hydrophilicity, with the efficiency of DNA restriction mapping separations. (4) To develop a model of DNA electrophoretic mobility in uncrosslinked polymer solutions, which allows prediction of the appropriate polymer molecular weight and concentration required to separate a given mixture of DNA restriction fragments, as a function of temperature and electric field strength. We also propose to develop a new sieving matrix coupled with a new high- frequency alternating crossed-field-gradient gel electrophoresis technique (HFAC-GE). The novel matrix is a polyacrylamide-based, interpenetrating polyampholyte gel with the gel fibers individually charged positively or negatively. The gel fibers will move in the alternating crossed-field- gradient thus altering the pore structure of the gel as the DNA migrates. This technique is designed to improve the resolution of DNA sequencing and to extend the range of restriction fragment mapping beyond that available with steady field methods.
| Sunada, W M; Blanch, H W (1998) Microscopy of DNA in dilute polymer solutions. Biotechnol Prog 14:766-72 |
| Sunada, W M; Blanch, H W (1998) A theory for the electrophoretic separation of DNA in polymer solutions. Electrophoresis 19:3128-36 |
