FLUORESCENCE POLARIZATION FOR DNA SEQUENCING

University Of South Carolina At Columbia, Columbia SC

Investigators

Linked publications & trials

Abstract

DESCRIPTION (Adapted from the Investigator's Abstract): 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 is 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.