NEW TECHNIQUE FOR HIGH-RESOLUTION DNA SIZING IN EPI-ILLUMINATION

We present a high-resolution DNA-sizing technique based on the princip les of flow cytometry, using a high numerical aperture objective and e pi-illumination. The new technique, designed for small fluorescing sam ples/particles (sub-micron diameter) suspended in a weakly fluorescent medium, makes use of an additional focus for high-precision particle localisation, This way, only those particles are considered that flow exactly through a well-defined volume. Results are presented for fluor escent beads, as well as for YOYO-stained plasmids containing 5,500 ba sepairs. The latter were measured with 6.2% resolution, setting a new limit to flow-based sizing of DNA. (C) 1998 Wiley-Liss, Inc.