High-throughput flow cytometric DNA fragment sizing

The rate of detection and sizing of individual fluorescently labeled DNA fr agments in conventional single-molecule now cytometry (SMFC) is limited by optical saturation, photon-counting statistics, and fragment overlap to sim ilar to 100 fragments/s. We have increased the detection rate for DNA fragm ent sizing in SMFC to similar to 2000 fragments/s by parallel imaging of th e fluorescence from individual DNA molecules, stained with a fluorescent in tercalating dye, as they passed through a planar sheet of excitation laser light, resulting in order of magnitude improvements in the measurement spee d and the sample throughout compared to conventional SMFC. Fluorescence bur sts were measured from a fM solution of DNA fragments ranging in size from 7 to 154 kilobase pairs. A data acquisition time of only a few seconds was sufficient to determine the DNA fragment size distribution. A linear relati onship between the number of detected photons per burst and the DNA fragmen t size was confirmed. Application of this parallel fluorescence imaging met hod will lead to improvements in the speed, throughput, and sensitivity of other types of flow-based analyses involving the study of single molecules, chromosomes, cells, etc.