DNA Fragment Sizing by High-Sensitivity Flow Cytometry: Applications in Bacterial Identification

High-sensitivity, single-molecule detection in flow is a paradigm that has been defined at Los Alamos over the last two decades. A recent focus has been on applications of single- molecule detection for DNA fragment sizing using a compact, low-power, highsensitivity flow cytometer (HSFCM). There are three key aspects of our approach that distinguish it from conventional flow cytometry and yield the high level of sensitivity that we achieve: a detector with high photon-detection efficiency, a small probe volume to reduce background noise, and slow flow to provide extended analyte dwell time in the probe volume. An additional factor for applications in DNA fragment sizing is a DNA stain with significant fluorescence enhancement when bound to double-stranded DNA, and low background fluorescence in the unbound state. DNA fragment sizing by HSFCM has important applications in bacterial species and strain identification, where it can replace the cumbersome and time-consuming pulsed-field gel electrophoresis (PFGE) approach routinely used by public health labs for bacterial identification. The revolutionary capability to interrogate single DNA molecules, as well as potentially other submicron-sized biological particles, in a high-sensitivity flow cytometer will provide new scientific insights into cellular and molecular biology and introduce high-sensitivity flow cytometry to a wide variety of new applications in biotechnology. Flow cytometry has enabled major advances in the biomedical sciences by providing rapid, quantitative, and sensitive multiparameter measurements of individual cells and subcellular particles such as chromosomes. This analysis of individual entities produces information on population heterogeneity that is not revealed in ensemble measurements and that allows more precise quantitation of distinct attributes than is possi ble when measurements are done in bulk. However, one limitation of conventional flow cytometry is the inability to measure submicron-sized particles or weakly fluorescent particles labeled with fewer than several hundred fluorophores, primarily as a result of insufficient detection sensitivity. A wide variety of important biological particles, molecules, and molecular assemblies fall into these categories. There have been many reports of bacterial measurement and characterization by conventional flow cytometry, dating back to 1947. In 1979, Steen developed a microscope-based system specifically for applications in microbiology. Many bacteria are large enough to generate a light-scatter signal, which is useful for their detection.