New procedures are needed to increase the readable length of a DNA sequencing ladder. Current procedures are limited primarily by resolution of the longer DNA fragments. The PI has developed a procedure for progressive increase in the resolution between DNA bands. No known limit to the achievable resolution exists. Resolution is progressively increased by back and forth motion of DNA; no increase in the length of the apparatus is required. Back and forth motion is induced by a pulsed electrical field; the pulses are orders of magnitude longer than previously used pulses. The resolution of DNA bands increases during both forward and reverse motion (bidirectional increase in resolution). Bidirectional increase in resolution is the cornerstone of the proposed work. Bidirectional increase in resolution for DNA sequencing is best achieved in a capillary format. The following are the specific aims: (a) Tests will be made of the effectiveness of bidirectional increase in resolution for increasing the readable length of a DNA sequencing ladder during capillary-based DNA sequencing. (b) Procedures for bidirectional increase in resolution will be optimized. The criterion for optimization will be reading of the longest possible DNA sequencing ladder in the shortest possible time. Preliminary experiments suggest that exploitation of anti- band broadening effects of pulsed fields will make a significant contribution. Achievement of the specific aims will improve both de novo sequencing and mutation.detection sequencing for all genomes.
| Griess, Gary A; Choi, Hyohoon; Basu, Arnab et al. (2002) Cyclic capillary electrophoresis. Electrophoresis 23:2610-7 |
| Griess, G A; Serwer, P (2001) Improving the length-fractionation of DNA during capillary electrophoresis. Electrophoresis 22:4320-7 |
