Viewing DNA Fragments

After the completion of a restriction reaction, a number of questions arise. Did the restriction enzyme cut the DNA? How many times was the DNA cut? What are the sizes of the resulting fragments? Gel electrophoresis provides us with a means of answering these questions.

Electrophoresis is a standard biochemical technique for separating molecules on the basis of their size and electrical charge. There are a number of different types of electrophoresis; to separate DNA molecules, gel electrophoresis is used. A porous gel is often made from agarose (a polysac-charide isolated from seaweed), which is melted in a buffer solution and poured into a plastic mold. As it cools, the agarose solidifies, making a gel that looks something like stiff gelatin.

Small indentions called wells are made at one end of the gel to hold solutions of DNA fragments (I Figure 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 (I Figure 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 determine the approximate size of the unknown fragments.

After electrophoresis, the DNA fragments are separated according to size (IFigure 18.4c). However, the DNA fragments are still too small to see; so the problem of visualizing the DNA needs to be addressed. Visualization can be accomplished in several ways. The simplest procedure is to stain the gel with a dye specific 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 fluoresces bright orange; so copies of each DNA fragment appear as a brilliant orange band (IFigure 18.4d). The original concentrated sample of purified 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 (32P) can be used as the substrate for DNA synthesis and will be incorporated into the newly synthesized DNA strand. In another method called end labeling, the bacteriophage enzyme polynucleotide kinase is used to

0 0

Post a comment