LARGE DNA FRAGMENT SIZING BY FLOW-CYTOMETRY - APPLICATION TO THE CHARACTERIZATION OF P1 ARTIFICIAL CHROMOSOME (PAC) CLONES

A flow cytometry-based, ultrasensitive fluorescence detection techniqu e is used to size individual DNA fragments up to 167 kb in length. App lication of this technology to the sizing of P1 artificial chromosomes (PACs) in both linear and supercoiled forms is described. It is demon strated that this method is well suited to characterizing PAC/BAC clon es and wilt be very useful for the analysis of large insert libraries. Fluorescence bursts are recorded as individual, dye stained DNA fragm ents pass through a low power, focused, continuous laser beam. The mag nitudes of the fluorescence bursts are linearly proportional to the le ngths of the DNA fragments. The histograms of the burst sizes are gene rated in <3 min with <1 pg of DNA. Results on linear fragments are con sistent with those obtained by pulsed-field gel electrophoresis. In co mparison with pulsed-field gel electrophoresis, sizing of large DNA fr agments by this approach is more accurate, much faster, requires much less DNA, and is independent of the DNA conformation.