Abstract

DNA fragment size distribution analysis is a ubiquitous measurement in molecular biology and is typically done using gel electrophoresis. Applications exist in public health, genomics, medical diagnostics, and forensics. We have fully demonstrated a flow cytometric approach for rapidly and accurately sizing DNA fragments.(1-5) Our work has been focused on bacterial species and strain identification. In our approach, a DNA sample of whole bacterial genome DNA is digested into a characteristic set of DNA fragments using a rare-cutting restriction endonuclease. The fragment set is stained with a fluorescent intercalating dye that binds stoichiometrically to the DNA such that the amount of dye incorporated is directly proportional to the fragment size [number of base pairs (bp)]. The large increase (~1000X) in the fluorescence quantum yield of the intercalating dye upon binding to the DNA makes it unnecessary remove unbound dye from the solution before analysis. The stained fragments are diluted to ~10⁻¹⁴ molar and introduced into an ultrasensitive flow cytometer developed in our laboratory. Fragments pass individually through the laser illuminated detection region of the flow cytometer, each fragment producing a fluorescence burst as it transits the laser beam. Fluorescence bursts from individual fragments are detected and recorded. A histogram of the individual burst sizes is generated that displays the distribution of fragment sizes in the sample (i.e. a DNA fingerprint). For large DNA fragments, our approach is more sensitive (femtograms vs. micrograms), faster (analysis time of five minutes vs. tens of hours), and more accurate (size uncertainty 2% vs. 10%) than pulsed-field gel electrophoresis, commonly used for these analyses.

© 2000 Optical Society of America