Preparation of Intact Chromosomal DNA from Gonyaulax polyedra Cells Ruptured in Hypo-osmotic Solution for PFGE Analysis

Algae > Volume 13(1); 1998 > Article

Algae 1998;13(1): 135-141.

Dong-Hee Lee, Jung-Sun Kim, So-Young Kim

Department of Biological Science and Research Institute of Life Science, Ewha Womans University

ABSTRACT

When intact chromosomal DNA of Gonyaulax polyedra, a marine dinoflagellate, needs to be prepared, removing cell wall is a critical step. Several digestive enzymes, e.g. pectinase, cellulase, and lysozyme, are known to be unable to dissolve outer cell layer of Gonyaulax. However, when G. polyhedra cells were mixed with a large volume of distilled water, the cells lysed spontaneously and the ruptured cells showed distinctive extrusion of cytoplasm, presumably at the girdle region. The rate and yield of cell lysis were increased when larger volume of distilled water mixed with the culture. While this process was relatively fast, completing within 5 min, it could be faster and gave higher yield in the presence of 5 or 10mM EDTA. In these cases, the extrusion was big, and dispersed. However, at higher concentrations of EDTA(e.g. 50 or 100 mM), the extrusion was much smaller, round and smooth appearance, but without distinctive cell rupturing. Typical ways of cell concentration technique, e.g. centrifugation, filtration through Nitex filter, filtration through filter paper with aspiration, chillig, or exposing to Liguinox detergent, cause motility changes as well as change of the sensitivity of the cells to osmotic stress. However, gentle shaking or slow filtration under illumination at ambient temperature didn't alter the motility nor sensitivity toward osmotic stress much. By combining the slow filtration method and osmotic lysis of the cells. the intact chromosomal DNA of Gonyaulax could be prepared successfully and analyzed by pulsed field gel electrophoresis (PFGE).

Key words: cell lysis, Dinoflagellates, Gonyaulax polyedra, osmotic stress, PFGE