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17.1
DNA Manipulation
    Learning Outcomes
  1. 1. Relate endogenous roles of enzymes to their recombinant DNA applications.
  2. 2. Explain why DNA fragments can be separated with gel electrophoresis.

The ability to directly isolate and manipulate genetic material was one of the most profound changes in the field of biology in the late 20th century. The construction of recombinant DNA molecules, that is, a single DNA molecule made from two different sources, began in the mid-1970s. The development of this technology, which has led to the entire field of biotechnology, is based on enzymes that can be used to manipulate DNA.

Restriction enzymes cleave DNA at specific sites

Enzymes called restriction endonucleases revolutionalized molecular biology because of their ability to cleave DNA at specific sites. As described in chapter 14, nucleases are enzymes that degrade DNA, and many were known prior to the isolation of the first restriction enzyme (HindII) in 1970. If a DNA sequence were a rope, then restriction enzymes would be a knife that always cut that rope into specific pieces.

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Discovery and significance of restriction endonucleases

This site-specific cleavage activity, long sought by molecular biologists, was discovered from basic research into why bacterial viruses can infect some cells but not others. This phenomenon was termed host restriction. The bacteria produce enzymes that can cleave the invading viral DNA at specific sequences. The host cells protect their own DNA from cleavage by modifying it at the cleavage sites; the restriction enzymes do not cleave that modified DNA. Since the initial discovery of these restriction endonucleases, hundreds more have been isolated that recognize and cleave different restriction sites.

The ability to cut DNA at specific places is significant in two ways: First, it allows physical maps to be constructed based on the positioning of cleavage sites for restriction enzymes. These restriction maps provide crucial data for identifying and working with DNA molecules.

Second, restriction endonuclease cleavage allows the creation of recombinant molecules. The ability to construct recombinant molecules is critical to research, because many steps in the process of cloning and manipulating DNA require the ability to combine molecules from different sources.

How restriction enzymes work

There are three types of restriction enzymes, but only type II cleaves at precise locations. Types I and III cleave with less precision and are not often used in cloning and manipulating DNA.

Type II enzymes allow creation of recombinant molecules; these enzymes recognize a specific DNA sequence, ranging from 4 bases to 12 bases, and cleave the DNA at a specific base within this sequence (figure 17.1).

Figure 17.1
Many restriction endonucleases produce DNA fragments with “sticky ends.”The restriction endonuclease EcoRI always cleaves the sequence 5′GAATTC3′ between G and A. Because the same sequence occurs on both strands, both are cut. However, the two sequences run in opposite directions on the two strands. As a result, single-stranded tails called “sticky ends” are produced that are complementary to each other. These complementary ends can then be joined to a fragment from another DNA that is cut with the same enzyme. These two molecules can then be joined by DNA ligase to produce a recombinant molecule.

Restriction Endonucleases

Steps in Cloning a Gene 2

The recognition sites for most type II enzymes are palindromes. A linguistic palindrome is a word or phrase that reads the same forward and in reverse, such as the sentence: “Madam I'm Adam.” The palindromic DNA sequence reads the same from 5′ to 3′ on one strand as it does on the complementary strand (see figure 17.1).

Given this kind of sequence, cutting the DNA at the same base on either strand can lead to staggered cuts that produce “sticky ends.” These short, unpaired sequences are the same for any DNA that is cut by this enzyme. Thus, these sticky ends allow DNAs from different sources to be easily joined together (see figure 17.1). While less common, some type II restriction enzymes, including PvuII, can cut both strands in the same position, producing blunt, not sticky, ends. Blunt cut ends can be joined with other blunt cut ends.

DNA ligase allows construction of recombinant molecules

Because the two ends of a DNA molecule cut by a type II restriction enzyme have complementary sequences, the pair can form a duplex. An enzyme is needed, however, to join the two fragments together to create a stable DNA molecule. The enzyme DNA ligase accomplishes this by catalyzing the formation of a phosphodiester bond between adjacent phosphate and hydroxyl groups of DNA nucleotides. The action of ligase is to seal nicks in one or both strands (see figure 17.1). This is the same enzyme that joins Okazaki fragments on the lagging strand during DNA replication (see chapter 14).

Gel electrophoresis separates DNA fragments

The fragments produced by restriction enzymes would not be of much use if we could not also easily separate them for analysis. The most common separation technique used is gel electrophoresis. This technique takes advantage of the negative charge on DNA molecules by using an electrical field to provide the force necessary to separate DNA molecules based on size.

The gel, which is made of either agarose or polyacrylamide and spread thinly on supporting material, provides a three-dimensional matrix that separates molecules based on size (figure 17.2). The gel is submerged in a buffer solution containing ions that can carry current and is subjected to an electrical field.

 
Figure 17.2
Gel electrophoresis.a. Three restriction enzymes are used to cut DNA into specific pieces depending on each enzyme's recognition sequence. b. The fragments are loaded into a gel (agarose or polyacrylamide), and an electrical current is applied. The DNA fragments migrate through the gel based on size, with larger ones moving more slowly. c. This results in a pattern of fragments separated based on size, with the smaller fragments migrating farther than larger ones. d. The fragments can be visualized by staining with the dye ethidium bromide. When the gel is exposed to UV light, the DNA with bound dye fluoresces, appearing as pink bands in the gel. In the photograph, one band of DNA has been excised from the gel for further analysis and can be seen glowing in the tube the technician holds.
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The strong negative charges from the phosphate groups in the DNA backbone cause it to migrate toward the positive pole (figure 17.2b). The gel acts as a sieve to separate DNA molecules based on size: The larger the molecule, the slower it will move through the gel matrix. Over a given period, smaller molecules migrate farther than larger ones. The DNA in gels can be visualized using a fluorescent dye that binds to DNA (figure 17.2c, d).

Electrophoresis is one of the most important methods in the toolbox of modern molecular biology, with uses ranging from DNA fingerprinting to DNA sequencing, both of which are described later on.

Transformation allows introduction of foreign DNA into E. coli.

The construction of recombinant molecules is the first step toward genetic engineering. It is also necessary to be able to reintroduce these molecules into cells. In chapter 14 you learned that Frederick Griffith demonstrated that genetic material could be transferred between bacterial cells. This process, called transformation, is a natural process in the cells that Griffith was studying.

Bacterial Transformation

The bacterium E. coli, used routinely in molecular biology laboratories, does not undergo natural transformation; but artificial transformation techniques have been developed to allow introduction of foreign DNA into E. coli. Through temperature shifts or an electical charge, the E. coli membrane becomes transiently permeable to the foreign DNA. In this way, recombinant molecules can be propagated in a cell that will make many copies of the constructed molecules.

In general, the introduction of DNA from an outside source into a cell is referred to as transformation. This process is important in E. coli for molecular cloning and the propagation of cloned DNA. Researchers also want to be able to reintroduce DNA into the original cells from which it was isolated. A transformed cell that can also be used to form all or part of an organism, is called a transgenic organism. Later in this chapter we explore the construction and uses of transgenic plants and animals.

17.1. DNA Manipulation
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