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Griffiths AJF, Gelbart WM, Miller JH, et al. Modern Genetic Analysis. New York: W. H. Freeman; 1999.

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Modern Genetic Analysis.

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Making Recombinant DNA

How does recombinant DNA technology work? The organism under study, which will be used to donate DNA for the analysis, is called the donor organism. The basic procedure is to extract and cut up DNA from a donor genome into fragments containing from one to several genes and allow these fragments to insert themselves individually into opened-up small autonomously replicating DNA molecules such as bacterial plasmids. These small circular molecules act as carriers, or vectors, for the DNA fragments. The vector molecules with their inserts are called recombinant DNA because they consist of novel combinations of DNA from the donor genome (which can be from any organism) with vector DNA from a completely different source (generally a bacterial plasmid or a virus). The recombinant DNA mixture is then used to transform bacterial cells, and it is common for single recombinant vector molecules to find their way into individual bacterial cells. Bacterial cells are plated and allowed to grow into colonies. An individual transformed cell with a single recombinant vector will divide into a colony with millions of cells, all carrying the same recombinant vector. Therefore an individual colony contains a very large population of identical DNA inserts, and this population is called a DNA clone. A great deal of the analysis of the cloned DNA fragment can be performed at the stage when it is in the bacterial host. Later, however, it is often desirable to reintroduce the cloned DNA back into cells of the original donor organism to carry out specific manipulations of genome structure and function. Hence the protocol is often as follows:

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MESSAGE

Cloning allows the amplification and recovery of a specific DNA segment from a large, complex DNA sample such as a genome.

Inasmuch as the donor DNA was cut into many different fragments, most colonies will carry a different recombinant DNA (that is, a different cloned insert). Therefore, the next step is to find a way to select the clone with the insert containing the specific gene in which we are interested. When this clone has been obtained, the DNA is isolated in bulk and the cloned gene of interest can be subjected to a variety of analyses, which we shall consider later in the chapter. Notice that the cloning method works because individual recombinant DNA molecules enter individual bacterial host cells, and then these cells do the job of amplifying the single molecules into large populations of molecules that can be treated like chemical reagents. Figure 10-1 on the following page gives a general outline of the approach.

Figure 10-1. Recombinant DNA technology enables individual fragments of DNA from any genome to be inserted into vector DNA molecules such as plasmids and individually amplified in bacteria.

Figure 10-1

Recombinant DNA technology enables individual fragments of DNA from any genome to be inserted into vector DNA molecules such as plasmids and individually amplified in bacteria. Each amplified fragment (more...)

The term recombinant DNA must be distinguished from the natural DNA recombinants that result from crossing-over between homologous chromosomes in both eukaryotes and prokaryotes. Recombinant DNA in the sense being used in this chapter is an unnatural union of DNAs from nonhomologous sources, usually from different organisms. Some geneticists prefer the alternative name chimeric DNA, after the mythological Greek monster Chimera. Down through the ages, the Chimera has stood as the symbol of an impossible biological union, a combination of parts of different animals. Likewise, recombinant DNA is a DNA chimera and would be impossible without the experimental manipulation that we call recombinant DNA technology.

Isolating DNA

The first step in making recombinant DNA is to isolate donor and vector DNA. General protocols for DNA isolation were available many decades before the advent of recombinant DNA technology. With the use of such methods, the bulk of DNA extracted from the donor will be nuclear genomic DNA in eukaryotes or the main genomic DNA in prokaryotes; these types are generally the ones required for analysis. The procedure used for obtaining vector DNA depends on the nature of the vector. Bacterial plasmids are commonly used vectors, and these plasmids must be purified away from the bacterial genomic DNA. A protocol for extracting plasmid DNA by ultracentrifugation is summarized in Figure 10-2 on page 303. Plasmid DNA forms a distinct band after ultracentrifugation in a cesium chloride density gradient containing ethidium bromide. The plasmid band is collected by punching a hole in the plastic centrifuge tube. Another protocol relies on the observation that, at a specific alkaline pH, bacterial genomic DNA denatures but plasmids do not. Subsequent neutralization precipitates the genomic DNA, but plasmids stay in solution. Phages such as λ also can be used as vectors for cloning DNA in bacterial systems. Phage DNA is isolated from a pure suspension of phages recovered from a phage lysate.

Figure 10-2. Plasmids such as those carrying genes for resistance to the antibiotic tetracycline (top left) can be separated from the bacterial chromosomal DNA.

Figure 10-2

Plasmids such as those carrying genes for resistance to the antibiotic tetracycline (top left) can be separated from the bacterial chromosomal DNA. Because differential binding of (more...)

Cutting DNA

The breakthrough that made recombinant DNA technology possible was the discovery and characterization of restriction enzymes. Restriction enzymes are produced by bacteria as a defense mechanism against phages. The enzymes act like scissors, cutting up the DNA of the phage and thereby inactivating it. Importantly, restriction enzymes do not cut randomly; rather, they cut at specific DNA target sequences, which is one of the key features that make them suitable for DNA manipulation. Any DNA molecule, from viruses to humans, contains restriction-enzyme target sites purely by chance and therefore may be cut into defined fragments of size suitable for cloning. Restriction sites are not relevant to the function of the organism, nor would they be cut in vivo, because most organisms do not have restriction enzymes.

Let’s look at an example: the restriction enzyme EcoRI (from E. coli) recognizes the following sixnucleotide-pair sequence in the DNA of any organism:

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This type of segment is called a DNA palindrome, which means that both strands have the same nucleotide sequence but in antiparallel orientation. Many different restriction enzymes recognize and cut specific palindromes. The enzyme EcoRI cuts within this sequence but in a pair of staggered cuts between the G and the A nucleotides:

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This staggered cut leaves a pair of identical single-stranded “sticky ends.” The ends are called sticky because they can hydrogen-bond (stick) to a complementary sequence. Figure 10-3 shows EcoRI making a single cut in a circular DNA molecule such as a plasmid: the cut opens up the circle, and the linear molecule formed has two sticky ends. Production of these sticky ends is another feature of restriction enzymes that makes them suitable for recombinant DNA technology. The principle is simply that, if two different DNA molecules are cut with the same restriction enzyme, both will produce fragments with the same complementary sticky ends, making it possible for DNA chimeras to form. Hence, if both vector DNA and donor DNA are cut with EcoRI, the sticky ends of the vector can bond to the sticky ends of a donor fragment when the two are mixed.

Figure 10-3. The restriction enzyme EcoRI cuts a circular DNA molecule bearing one target sequence, resulting in a linear molecule with single-stranded sticky ends.

Figure 10-3

The restriction enzyme EcoRI cuts a circular DNA molecule bearing one target sequence, resulting in a linear molecule with single-stranded sticky ends.

MESSAGE

Restriction enzymes have two properties useful in recombinant DNA technology. First, they cut DNA into fragments of a size suitable for cloning. Second, many restriction enzymes make staggered cuts generating single-stranded sticky ends conducive to the formation of recombinant DNA.

Dozens of restriction enzymes with different sequence specificities have now been identified, some of which are shown in Table 10-1. You will notice that all the target sequences are palindromes, but, like EcoRI, some enzymes make staggered cuts, whereas others make flush cuts. Even flush cuts, which lack sticky ends, can be used for making recombinant DNA.

Table 10-1. Recognition, Cleavage, and Modification Sites of Various Restriction Enzymes.

Table 10-1

Recognition, Cleavage, and Modification Sites of Various Restriction Enzymes.

DNA can also be cut by mechanical shearing. For example, agitating DNA in a blender will break up the long chromosome-sized molecules into flush-ended clonable segments.

Joining DNA

Most commonly, both donor DNA and vector DNA are digested with the use of a restriction enzyme that produces sticky ends and then mixed in a test tube to allow the sticky ends of vector and donor DNA to bind to each other and form recombinant molecules. Figure 10-4a shows a plasmid vector that carries a single EcoRI restriction site, so digestion with the restriction enzyme EcoRI converts the circular DNA into a linear molecule with sticky ends. Donor DNA from any other source (say, Drosophila) also is treated with the EcoRI enzyme to produce a population of fragments carrying the same sticky ends. When the two populations are mixed, DNA fragments from the two sources can unite, because double helices form between their sticky ends. There are many opened-up vector molecules in the solution, and many different EcoRI fragments of donor DNA. Therefore a diverse array of vectors carrying different donor inserts will be produced. At this stage, although sticky ends have united to generate a population of chimeric molecules, the sugar-phosphate backbones are still not complete at two positions at each junction. However, the backbones can be sealed by the addition of the enzyme DNA ligase, which creates phosphodiester bonds at the junctions (Figure 10-4b). Certain ligases are even capable of joining DNA fragments with blunt-cut ends.

Figure 10-4. Method for generating a chimeric DNA plasmid containing genes derived from foreign DNA.

Figure 10-4

Method for generating a chimeric DNA plasmid containing genes derived from foreign DNA. (From S. N. Cohen, “The Manipulation of Genes.” Copyright © 1975 by Scientific (more...)

Amplifying Recombinant DNA

The ligated recombinant DNA enters a bacterial cell by transformation. After it is in the host cell, the plasmid vector is able to replicate because plasmids normally have a replication origin. However, now that the donor DNA insert is part of its length, the donor DNA is automatically replicated along with the vector. Each recombinant plasmid that enters a cell will form multiple copies of itself in that cell. Subsequently, many cycles of cell division will occur, and the recombinant vectors will undergo more rounds of replication. The resulting colony of bacteria will contain billions of copies of the single donor DNA insert. This set of amplified copies of the single donor DNA fragment is the DNA clone (Figure 10-5 on the following page).

Figure 10-5. How amplification works.

Figure 10-5

How amplification works. Restriction enzyme treatment of donor DNA and vector allows insertion of single fragments into vectors. A single vector enters a bacterial host, where replication (more...)

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 1999, W. H. Freeman and Company.
Bookshelf ID: NBK21407

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