Small indentions called wells are made at one end of the gel to hold solutions

Small indentions called wells are made at one end of

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Small indentions called wells are made at one end of the gel to hold solutions of DNA fragments ( F IGURE 18.4a) , and an electrical current is passed through the gel. Because the phosphate of each DNA nucleotide carries a negative charge, the DNA fragments migrate toward the positive end of the gel ( F IGURE 18.4b) . In this migration, the gel acts as a sieve; as the DNA molecules migrate toward the positive pole, they move through the pores between the gel particles. Small DNA fragments migrate more rapidly than do large ones and, with time, the fragments separate on the basis of their size. The distance that each fragment migrates depends on its size. Typically, DNA fragments of known length (a marker sample) are placed in another well. By comparing the migration distance of the unknown fragments with the distance traveled by the marker fragments, one can deter- mine the approximate size of the unknown fragments. After electrophoresis, the DNA fragments are separated according to size ( F IGURE 18.4c) . However, the DNA frag- ments are still too small to see; so the problem of visualiz- ing the DNA needs to be addressed. Visualization can be accomplished in several ways. The simplest procedure is to stain the gel with a dye speci fi c for nucleic acids, such as ethidium bromide, which wedges itself tightly (intercalates) between the bases of DNA. When exposed to UV light, ethid- ium bromide fl uoresces bright orange; so copies of each DNA fragment appear as a brilliant orange band ( F IGURE 18.4d) . The original concentrated sample of puri fi ed DNA contained millions of copies of a DNA molecule, and thus each band represents millions of copies of identical DNA fragments. Alternatively, DNA fragments can be visualized by adding a radioactive or chemical label to the DNA before it is placed in the gel. Nucleotides with radioactively labeled phosphate ( 32 P) can be used as the substrate for DNA syn- thesis and will be incorporated into the newly synthesized DNA strand. In another method called end labeling, the bacteriophage enzyme polynucleotide kinase is used to
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514 Chapter 18 transfer a single 32 P to the 5 end of each DNA strand. Radioactively labeled DNA can be detected with a technique called autoradiography (see Figure 10.4), in which a piece of X-ray fi lm is placed on top of the gel. Radiation from the labeled DNA exposes the fi lm, just as light exposes photo- graphic fi lm in a camera. The developed autoradiograph gives a picture of the fragments in the gel; each DNA frag- ment appears as a dark band on the fi lm. Chemical labels can be detected by adding antibodies or other substances that carry a dye and will attach to the relevant DNA, which can be visualized directly. Gel electrophoresis is used widely in recombinant DNA technology; it is often employed when there is a need to determine the number or size of DNA fragments or to iso- late DNA fragments by size. For example, to determine the number and location of Bam HI restriction sites in a plas- mid, we might cut the plasmid by using the Bam HI restric-
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