Preparation and Separation of Intact Chromosomes of Vertebrates by One-Dimensional Pulsed-Field Gel Electrophoresis (ODPFGE)

This novel way of preparing chromosomes for pulsed-field gel electrophoresis (PFGE) takes advantage of the fact that the whole chromosome population is synchronized in metaphase. This is a very important step toward their intact separation by PFGE; for instance, a standard preparation as used for digestion with rarecutter enzymes shows a different pattern of resolution, characterized by diffuse bands and nonspecific migration (see Chapter 7 ). Here, vertebrate chromosomal DNA was prepared by a modified chromosome isolation procedure for flow cytometry (1 ). This procedure involves lysis of cells (blocked in metaphase by colcemide) with digitonine in the presence of spermidine and spermine as described below. The structural integrity of metaphase-blocked chromosomes is given by the presence of spermine and spermidine, which act as chromosomal morphology stabilizers. The digestion with proteinase K is carried out as described before in order to eliminate chromosomal proteins. The pulse parameters for separating intact chicken microchromosomes by one-dimensional pulsed-field gel electrophoresis (ODPFGE) are also given.