Gel Electrophoresis Allows Separation of Vector DNA from Cloned Fragments

In order to manipulate or sequence a cloned DNA fragment, it first must be separated from the vector DNA. This can be accomplished by cutting the recombinant DNA clone with the same restriction enzyme used to produce the recombinant vectors originally. The cloned DNA and vector DNA then are subjected to gel electrophoresis, a powerful method for separating DNA molecules of different size.

Near neutral pH, DNA molecules carry a large negative charge and therefore move toward the positive electrode during gel electrophoresis. Because the gel matrix restricts random diffusion of the molecules, molecules of the same length migrate together as a band whose width equals that of the well into which the original DNA mixture was placed at the start of the electrophoretic run. Smaller molecules move through the gel matrix more readily than larger molecules, so that molecules of different length migrate as distinct bands (Figure 9-21). DNA molecules composed of up to «2000 nucleotides usually are separated electrophoretically on polyacrylamide gels, and molecules from about 200 nucleotides to more than 20 kb on agarose gels.

A common method for visualizing separated DNA bands on a gel is to incubate the gel in a solution containing the fluorescent dye ethidium bromide. This planar molecule binds to DNA by intercalating between the base pairs. Binding concentrates ethidium in the DNA and also increases its intrinsic fluorescence. As a result, when the gel is illuminated with ultraviolet light, the regions of the gel containing DNA fluoresce much more brightly than the regions of the gel without DNA.

Once a cloned DNA fragment, especially a long one, has been separated from vector DNA, it often is treated with various restriction enzymes to yield smaller fragments. After separation by gel electrophoresis, all or some of these smaller fragments can be ligated individually into a plasmid vector and cloned in E. coli by the usual procedure. This process, known as subcloning, is an important step in rearranging parts of genes into useful new configurations. For instance, an investigator who wants to change the conditions under which a gene is expressed might use subcloning to replace the normal promoter associated with a cloned gene with a DNA segment containing a different promoter. Subcloning also can be used to obtain cloned DNA fragments that are of an appropriate length for determining the nucleotide sequence.

DNA restriction fragments

DNA restriction fragments

Place mixture in the well of an agarose or polyacrylamide gel. Apply electric field

M EXPERIMENTAL FIGURE 9-21 Gel electrophoresis separates DNA molecules of different lengths. A gel is prepared by pouring a liquid containing either melted agarose or unpolymerized acrylamide between two glass plates a few millimeters apart. As the agarose solidifies or the acrylamide polymerizes into polyacrylamide, a gel matrix (orange ovals) forms consisting of long, tangled chains of polymers. The dimensions of the interconnecting channels, or pores, depend on the concentration of the agarose or acrylamide used to form the gel. The separated bands can be visualized by autoradiography (if the fragments are radiolabeled) or by addition of a fluorescent dye (e.g., ethidium bromide) that binds to DNA.

Well

Gel particle

Gel particle

Lower Your Cholesterol In Just 33 Days

Lower Your Cholesterol In Just 33 Days

Discover secrets, myths, truths, lies and strategies for dealing effectively with cholesterol, now and forever! Uncover techniques, remedies and alternative for lowering your cholesterol quickly and significantly in just ONE MONTH! Find insights into the screenings, meanings and numbers involved in lowering cholesterol and the implications, consideration it has for your lifestyle and future!

Get My Free Ebook


Post a comment