The goal of the Phase II grant is to develop a DNA sequencing matrix for capillary electrophoresis that achieves very long read lengths (1000 bases) in a semi-automatic, high-throughput environment. We will use the analysis tools developed in Phase I to conduct an advanced investigation of the behavior of linear polymers as sieving matrices for DNA sequencing. We will concentrate on identification and elimination of the factors that contribute to peak broadening. We will investigate the effect of the molecular mass distribution of the sieving polymers on selectivity in both Ogston and reputation modes, as well as the effect of chemical composition of the polymers on both selectivity and separation efficiency. Further, we will optimize the composition of the background electrolyte to improve both selectivity and separation efficiency. This effort will lead to a sieving matrix formulation that supports long sequencing readlengths in a semi-automatic, high-throughput sequencing system.
Current commercial capillary sequencers produce high-accuracy sequencing read lengths averaging about 450 bases and rarely exceeding 600 bases. This work has the potential to double the average read length. This would reduce the cost of EST sequencing by half, and would be more significant for genome sequencing, since long read lengths greatly facilitate sequence assembly and closure. The resulting matrix could be quickly supplied for commercial use on the MegaBACE 1000 DNA Sequencing System.
| Dolnik, V; Gurske, W A; Padua, A (2001) Sieving matrices in capillary electrophoresis: inflection slope and double reciprocal plot. Electrophoresis 22:692-8 |
| Dolnik, V; Gurske, W A; Padua, A (2001) Galactomannans as a sieving matrix in capillary electrophoresis. Electrophoresis 22:707-19 |
| Horvath, J; Dolnik, V (2001) Polymer wall coatings for capillary electrophoresis. Electrophoresis 22:644-55 |
| Dolnik, V; Liu, S; Jovanovich, S (2000) Capillary electrophoresis on microchip. Electrophoresis 21:41-54 |
| Dolnik, V; Gurske, W A (1999) Capillary electrophoresis in sieving matrices: selectivity per base, mobility slope, and inflection slope. Electrophoresis 20:3373-80 |
| Dolnik, V (1999) DNA sequencing by capillary electrophoresis (review). J Biochem Biophys Methods 41:103-19 |
