High speed capillary gel electrophoresis made rapid DNA sequencing possible in the late 1980's. In the long term, the Human Genome Project will require the highest possible rates of sequencing. The goal of this project is to accelerate and improve the precision of single-lane DNA sequencing by refining the methodology used to detect the eluents from the capillary electrophoresis. Today, the dominant detection method for DNA fragments eluting from the capillary gel is via laser-induced fluorescence. Conventional color-discrimination schemes require wavelength selectivity in either the absorption or the emission spectrum of the labeling dyes. And current technology limits the number of dyes that can be effectively distinguished. The most obvious means of increasing selectivity also reduces sensitivity, and this trade-off is unacceptable due to the low signal levels that must be measured during sequencing.
The specific aim i s to show that fluorescence polarization can increase selectivity of dyes while simultaneously maintaining sensitivity. Fluorescence polarization is normally zero for small molecules in fluid solution, but the investigators will show that both viscous solvents and micellar media slow rotational depolarization substantially for DNA fragments, even in fluid solution. This will permit polarization to be measured accurately and reliably during sequencing.
| Yan, Y; Myrick, M L (2001) Identification of nucleotides with identical fluorescent labels based on fluorescence polarization in surfactant solutions. Anal Chem 73:4508-13 |
