Chantal Turmel

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.

Preparation, Manipulation, and Pulse Strategy for One-Dimensional Pulsed-Field Gel Electrophoresis (ODPFGE).

The preparation and manipulation of very large DNA molecules for pulsed-field gel electrophoresis (PFGE) requires more care than normally used for smaller molecules. The general protocol used is the preparation of DNA directly in solid agarose blocks (plugs) or beads. Intact cells are encapsidated in agarose (plugs or beads) and are treated with different combinations of enzymes and detergent to remove cell walls, membranes, RNA, proteins, and other materials in order to obtain naked DNA. This is possible because detergent and enzymes can diffuse by Brownian motion through the agarose pores of the plug, whereas the large pieces of DNA cannot and remain sequestered inside the plug. Here, we give detailed protocols of preparation for yeast and mammalian DNA. In addition, we describe the power supplies, switchers, gels, and gel trays required for one-dimensional pulsed-field gel electrophoresis (ODPFGE), as well as the pulse strategies used for the separations.

Elastic Bag Model of One-Dimensional Pulsed-Field Gel Electrophoresis (ODPFGE)

Gel electrophoresis is one of the most common techniques used in molecular biology for the separation of DNA molecules. Conventional gel electrophoresis (using a static electric field) does not permit separation of DNA fragments larger than 30-50 kbp (1) as shown in Fig. 1A of Chapter 7 . This is a surprising result as one would think that larger molecules would suffer a larger retardation, and separation over any size range would be possible. The inability to separate is related to the molecular conformation of a polyelectrolyte, such as DNA, migrating in a disordered medium, such as a gel, under the influence of a static electric field. During continuous field electrophoresis, the larger DNA fragments tend to orient and stretch in the field direction because they migrate in a one-dimensional fashion between the gel fibers (2-4). When this orientation is negligible, e.g., for smaller molecules or for very low field intensities, they maintain a three-dimensional random-walk conformation intertwined with the gel fibers during migration, and experience a retardation that is proportional to the mol size. However, when the orientation becomes large, the molecules become stretched and migrate essentially linearly along the field direction (5). The electrophoretic mobility then becomes independent of the mol size and no separation of molecules of different sizes is possible (Fig. 1A of Chapter 7 ). Physically, this is a consequence of the fact that for long molecules stretched and oriented in the field direction, both the electrical force on the molecule and the average friction opposing the forward motion are proportional to the length. It follows that the velocity, which is the ratio of these two quantities, depends only on the force per unit length and is independent of the actual mol length. This explains why a plateau of length-independent mobility is reached in a continuous electric field (Fig. 2 of Chapter 7 ).

Preparation, manipulation, and pulse strategy for one-dimensional pulsed-field gel electrophoresis (ODPFGE)

The underlying principles for zero-integrated-field electrophoresis (ZIFE) pulses and more general forward-biased pulse schemes are reviewed for one-dimensional pulsed-field gel electrophoresis (ODPFGE) separations of large DNA molecules. Detailed descriptions of materials, preparation protocols, hardware requirements, and procedures are given. A variety of gel pictures for known yeast DNA markers are shown.

Sequencing using pulsed field and image reconstruction

The use of pulsed fields in a standard manual sequencing set-up results in the separation of over 2000 bases on a single gel. However visual reading of the sequence from a film is not possible for more than about 800 - 900 bases under the best conditions. The use of image reconstruction and analysis techniques allows the reading of the M13 mp18 sequence up to about 1600 - 1700 bases, and individual bands can be identified above 2000 bases.