Gel Electrophoresis of DNA

Perhaps the most widely used physical method in all of molecular biology is gel electrophoresis. This technique separates and purifies fragments of DNA or RNA as well as proteins. The basic idea of electrophoresis is to separate the molecules based on their intrinsic electrical charge. Electrically positive charges attract negative charges and repel other positive charges. Conversely, negative charges attract positive charges and repel other negative charges. Two electrodes, one positive and the other negative, are connected up to a high voltage source. Positively charged molecules move towards the negative electrode and negatively charged molecules move towards the positive electrode (Fig. 21.03).

Since DNA carries a negative charge on each of the many phosphate groups making up its backbone, it will move towards the positive electrode during elec-trophoresis. The bigger a molecule, the more force required to move it. However, the longer a DNA molecule, the more negative charges it has. In practice, these two factors cancel out because all fragments of DNA have the same number of charges per unit length. Consequently, DNA molecules in free solution will all move toward the positive electrode at the same speed, irrespective of their molecular weights.

Electrophoresis of DNA is usually used to separate the DNA into different sizes. For example, scientists will often isolate DNA from bacteria. In addition to the chromosome, bacteria often contain plasmids; however, gel electrophoresis will separate the two different sized molecules of DNA. The gel that separates the fragments consists of a matrix of cross-linked polymer chains. Most DNA is separated using agarose gel electrophoresis. Agarose is a polysaccharide extracted from seaweed. When agarose and water are mixed and boiled, the agarose melts into a homogeneous solution. As the solution cools, it gels to form a meshwork, which has small pores or openings filled with water. The cooled gel looks much like a very concentrated mixture of gelatin without the food coloring. The pore size of agarose is suitable for separating nucleic acid polymers consisting of several hundred nucleotides or longer. Shorter fragments of DNA as well as proteins are usually separated on gels made of polyacry-

agarose A polysaccharide from seaweed that is used to form gels for separating nucleic acids by electrophoresis agarose gel electrophoresis Technique for separation of nucleic acid molecules by passing an electric current through a gel made of agarose electrophoresis Movement of charged molecules due to an electric field. Used to separate and purify nucleic acids and proteins gel electrophoresis Electrophoresis of charged molecules through a gel meshwork in order to sort them by size polyacrylamide Polymer used in separation of proteins or very small nucleic acid molecules by gel electrophoresis

FIGURE 21.03 Principle of Electrophoresis

Creating an electrical field in a solution of positive and negatively charged ions allows the isolation of the ions with different charges. Since DNA has a negative charge due to its phosphate backbone, electrophoresis will isolate the negatively charged DNA from other components.

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FIGURE 21.03 Principle of Electrophoresis

Creating an electrical field in a solution of positive and negatively charged ions allows the isolation of the ions with different charges. Since DNA has a negative charge due to its phosphate backbone, electrophoresis will isolate the negatively charged DNA from other components.

FIGURE 21.04 Agarose Gel Electrophoresis of DNA

Agarose gel electrophoresis separates fragments of DNA by size. Negatively charged DNA molecules are attracted to the positive electrode. As the DNA migrates, the fragments of DNA are hindered by the cross-linked agarose meshwork. The smaller the piece of DNA, the less likely it will be slowed down. Therefore, smaller fragments of DNA migrate faster.

FIGURE 21.04 Agarose Gel Electrophoresis of DNA

Agarose gel electrophoresis separates fragments of DNA by size. Negatively charged DNA molecules are attracted to the positive electrode. As the DNA migrates, the fragments of DNA are hindered by the cross-linked agarose meshwork. The smaller the piece of DNA, the less likely it will be slowed down. Therefore, smaller fragments of DNA migrate faster.

DNA can be detected by radioactive labeling or by staining with a dye such as ethidium bromide.

â– amide. The meshwork formed by this polymer has smaller pores than agarose polymers. The samples of protein or DNA are loaded into a slot or sample well at the end of the gel closest to the negative electrode. DNA molecules move through the gel away from the negative electrode and towards the positive electrode. As the DNA molecules move through the gel they are hindered by the meshwork of fibers that make up the gel. The larger molecules find it more difficult to squeeze through the gaps but the smaller ones are slowed down much less. The result is that the DNA fragments separate in order of size (Fig. 21.04). In our example, the rings of plasmid DNA will move farther in the gel than the chromosome.

Agarose gels are normally square slabs that allow multiple samples to be run side by side. Because DNA is naturally colorless, some way of visualizing the DNA after running the gel is needed. The DNA may be radioactively labeled and detected by autoradiography, as explained below. Alternatively, the gel can be stained with ethidium bromide, which binds tightly and specifically to DNA or RNA. Ethidium bromide ethidium bromide A stain that specifically binds to DNA or RNA and appears orange if viewed under ultraviolet light

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