The technique of pulsed-field agarose-gel electrophoresis was used to separate the 16 different chromosomes of the yeast species Saccharomyces cerevisiae, which range in size from 220,000 to 2.5 million nucleotide pairs. The DNA was stained as in (B). DNA molecules as large as 107 nucleotide pairs can be separated in this way. (B, from U. Albrecht et al., J. Gen. Virol. 75:3353-3363, 1994; C, courtesy of Leander Lauffer and Peter Walter; D, from D. Vollrath and R.W. Davis, Nucleic Acids Res. 15:7865–7876, 1987. With permission from Oxford University Press.) 2.5 4 million 15 7 950,000 6.5 4.3 14 10 11 610,000 2.3 2 3 6 1 220,000 (A) slab of agarose gel (B) (C) (D)
Purified DNA Molecules Can Be Specifically Labeled with Radioisotopes or Chemical Markers in vitro
The DNA polymerases that synthesize and repair DNA (discussed in Chapter 5) have become important tools in experimentally manipulating DNA. Because they synthesize sequences complementary to an existing DNA molecule, they are often used in the test tube to create exact copies of existing DNA molecules. The copies can include specially modified nucleotides (Figure 8–26). To synthesize DNA in this way, the DNA polymerase is presented with a template and a pool of nucleotide precursors that contain the modification. As long as the polymerase can use these precursors, it automatically makes new, modified molecules that match the sequence of the template. Modified DNA molecules have many uses. DNA labeled with the radioisotope 32P can be detected following gel electrophoresis by placing the gel next to a piece of photographic film (see Figure 8–25C). The 32P atoms emit β particles which expose the film, producing a visible record of every band on the gel. Alternatively, the gel can be scanned by a detector that measures the β emissions directly. Other types of modified DNA, such as that labeled by digoxigenin (see Figure 8–26B), are useful for visualizing DNA molecules in whole cells, a topic we discuss later in this chapter.
Any DNA fragment can be cloned. In molecular biology, the term DNA cloning is used in two senses. It literally refers to the act of making many identical copies (typically billions) of a DNA molecule—the amplification of a particular DNA sequence. However, the term also describes the isolation of a particular stretch of DNA (often a particular gene) from the rest of the cell’s genome; the same term purifed fragment of duplex DNA
DNA polymerase incorporates 32P nucleotides, resulting in a population of radiolabeled DNA molecules that contain sequences from both strands
Figure 8–26 Methods for labeling DNA molecules in vitro. (A) A purified DNA polymerase enzyme can incorporate radiolabeled nucleotides as it synthesizes new DNA molecules. In this way, radiolabeled versions of any DNA sequence can be prepared in the laboratory. (B) The method in (A) is also used to produce nonradioactive DNA molecules that carry a specific chemical marker that can be detected with an appropriate antibody. The base on the nucleoside triphosphate shown is an analog of thymine, in which the methyl group on T has been replaced by a spacer arm linked to the plant steroid digoxigenin. An anti-digoxigenin antibody coupled to a visible marker such as a fluorescent dye is then used to visualize the DNA. Other chemical labels, such as biotin, can be attached to nucleotides and used in the same way. The only requirements are that the modified nucleotides properly base-pair and appear “normal” to the DNA polymerase.