Major current limitations of large-scale genome sequencing can be overcome by achieving the following objectives: (1) increasing the readable length of a DNA sequencing ladder, and (2) incorporating error-flagging-correcting into the process of analyzing a DNA sequencing ladder. The proposed research will achieve both these objectives by use of capillary DNA sequencing. The first objective will be achieved by increasing the effective length of the path of electrophoresis, without increasing the physical length of the capillary. A DNA sequencing ladder will be analyzed by use of several stages (analysis-stages) of conventional capillary electrophoresis. An enhancement-stage will be inserted between each neighboring pair of analysis-stages. During an enhancement-stage, a pulsed electrical field is used to improve the resolution of the unanalyzed portion of the DNA sequencing ladder, while the DNA molecules migrate slightly in a direction opposite to the direction of an analysis-stage. This experimentally-proven strategy (called cyclic capillary electrophoresis) length-resolves DNA fragments that are compressed during an uninterrupted constant field electrophoresis. DNA peaks do not broaden during cyclic capillary electrophoresis. The cyclic character opens the possibility of error-flagging by analyzing the same DNA sequencing fragments more than once. The current data suggest that dramatic advances remain to be made. Needed equipment-software has recently been developed by the PI and his collaborators. Thus, the specific aims are the following: (1) we will develop optimized procedures for using cyclic capillary electrophoresis to increase the readable length of a DNA sequencing ladder. We will test the effects of embedding additional pulses within the pulses of enhancement-stages. (2) We will investigate the use of cyclic electrophoresis for error flagging-correcting during capillary DNA sequencing. (3) We will develop a user-friendly interface for integrating cyclic electrophoresis with current procedures of DNA sequencing. The interface will provide access to different adaptations of cyclic capillary electrophoresis. Each adaptation will help solve a current genomic problem, for example, either closing of a partially assembled genome sequence or performing high accuracy analysis of mutations.
| Serwer, Philip; Hayes, Shirley J; Thomas, Julie A et al. (2007) Rapid determination of genomic DNA length for new bacteriophages. Electrophoresis 28:1896-902 |
| Griess, Gary A; Hardies, Stephen C; Serwer, Philip (2005) Temperature-dependence of preconditioning for lengthened capillary DNA sequencing. Electrophoresis 26:4440-8 |
| Griess, Gary A; Hardies, Stephen C; Serwer, Philip (2005) Matrix conditioning for lengthened capillary DNA sequencing. Electrophoresis 26:102-11 |
