Electrophoresis through a Gel Separates DNA and RNA Molecules According to Size
We begin by discussing the separation of DNA and RNA molecules by the technique of gel electrophoresis. Linear DNA molecules separate according to size when subject to an electric field through a gel matrix, an inert, jellodike porous material, Because DNA is negatively charged, when subject to an electrical field in this way, it migrates through the gel toward the positive pole (Figure 20-]). DMA molecules are flexible and occupy an effective volume. Pores m the gel matrix sieve the DNA molecules according to this volume; large molecules migrate metre slowly through the gel because they have a larger effective volume than do smaller DNAs, and thus have more difficulty passing through the interstices of the gel. This means that once the gels have been "run" for a given time, molecules of different sizes are separated because they have moved different distances through the gel.
After electrophoresis is complete, the DNA molecules can be visualized by staining the gel with fluorescent dyes, such as ethidium, which binds to DNA and intercalates between the stacked bases (see Figure G-Za). Each band reveals the presence of a population of DNA molecules of a specific size.
Two alternative kinds of gel matrices are used: polyacrylamide and agarose. Polyacrylamide has high resolving capability but can
FIGURE 20-1 DNA separation by gel electrophoresis. Tile figure shows a gel from the Side in cross-section. Thus the "welt" into which the DNA mixture is loaded onto the gel is indicated at the left, at the head of the gel. That ts also the end at which the cathode of the olec trie field is located, the «node being at the fool of the gel. As a result the DNA fragments, which are negatively charged, move through the gel from the head to the foot The distance they travel is inversely related to the size of the DNA fragment, as shown, (Source: Adapted from Witklos D.A. and Freyet CA 2003. DNA science: A first course, 2nd edition, p. 114. Cold Spting Harbor Laboratory Press, Cold Spnng harbor, NY.)
electrophoresis chamber buffer solution DNA fragments agarose gel
electrode electrode e ©
electrode small DNA fragments move further through the gel than large fragments electrode ©
separate DNAs only over a narrow size range. Thus, electrophoresis through polyacrylamide can resolve DNAs that differ from each other in size by as little as a single base pah but only with molecules of up to several hundred base pairs. Agarose has less resolving power than polyacrylamide but can separate from one another DNA molecules of up to tens, and even hundreds, of kilobases.
Very long DNAs are unable to penetrate the pores even in agarose. Instead, they snake their way through the matrix with one end leading tlie way and the other end trailing from behind. As a consequence, DNA molecules above a certain size (30 to 50 kb} migrate to a similar extent and so cannot readily he resolved. These very long DNAs can, however, be resolved from one another if the electric held is applied in pulses that are oriented orthogonally to each other. This technique is known as pulsed-field gel electrophoresis (Figure 20-2). Each time the orientation of the electric held changes, the DNA molecule, which is snaking its way through the gel, must reorient to the direction of the new held. The larger the DNA, the longer it takes to reorient. Fulsed-field gel electrophoresis can be used to determine the size of entire bacterial chromosomes and chromosomes of lower eukaryotes, such as fungi. That is, molecules of up to several Mb in length.
Electrophoresis separates DNA molecules, not only according to their molecular weight, but also according to their shape and topological properties. A circular DNA molecule that is relaxed or nicked migrates more slowly than does a linear molecule of equal mass. Also, as we have seen, supercoiled DNAs, which are compact and have a small effective volume, migrate more rapidly during electrophoresis than do less supercoiled or relaxed circular DNAs of equal mass (Chapter 6, Figure 6-26).
Electrophoresis is used to separate RNAs as well. Linear double-stranded DNAs have a uniform secondary structure, and their rate of migration during electrophoresis is proportional to their molecular weight, kike DNAs, RNAs have a uniform negative charge. But RNA molecules are usually single-stranded and have, as we have seen (Chapter 6), extensive secondary and tertiary structure, which influences their electrophoretic mobility. To deal with this, RNAs can be treated with reagents, such as glyoxaL, that react with the RNA in such a way as to prevent the formation of base pairs (glyoxa! forms ad ducts with amino groups in the bases, thereby preventing base-pairing]. Glyoxylated RNAs are unable to form secondary or tertiary structures and hence migrate with a mobility that is approximately proportional to molecular weight. As we will see in a later section, electrophoresis is used in a similar way to separate proteins on the basis of their size,
Nucleic Acids 649 electrodes y
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