The first application of recombinant DNA technology was the introduction of foreign DNA fragments into the cells of bacteria in the early 1970s. The method is simple and straightforward, and remains one of the mainstays of modern molecular research. It can be used to generate a large quantity of a protein for study or therapeutic use.

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The method requires a fragment of double-stranded DNA that serves as the donor. The donor fragment may be a protein-coding gene, a regulatory part of a gene, or any DNA segment of interest. If you are interested in generating bacteria that could produce human insulin, you might use the coding region of the human insulin gene as your donor DNA molecule. Also required is a vector sequence into which the donor fragment is to be inserted. The vector is the carrier of the donor fragment, and it must have the ability to be maintained in bacterial cells. A frequently used vector is a bacterial plasmid, a small circular molecule of DNA found naturally in certain bacteria that can replicate when the bacterial genomic DNA replicates and be transmitted to the daughter bacterial cells when the parental cell divides. Many naturally occurring plasmids have been modified by genetic engineering to make them suitable for use as vectors in recombinant DNA technology.

A common method for producing recombinant DNA is shown in Fig. 12.19. In order to make sure donor DNA can be fused with vector DNA, both pieces are cut with the same restriction enzyme so they both have the same overhangs. In the example shown in Fig. 12.19, the donor DNA is a fragment produced by digestion of genomic DNA with the restriction enzyme EcoRI, resulting in four-nucleotide 5′ overhangs at the fragment ends. The vector is a circular plasmid that contains a single EcoRI cleavage site, so digestion of the vector with EcoRI opens the circle with a single cut that also has four-nucleotide 5′ overhangs. Note that the overhangs on the donor fragment and the vector are complementary, allowing the ends of the donor fragment to renature with the ends of the opened vector when the two molecules are mixed. Once this renaturation has taken place, the ends of the donor fragment and the vector are covalently joined by DNA ligase. The joining of the donor DNA to the vector creates the recombinant DNA molecule.

The next step in the procedure is transformation, in which the recombinant DNA is mixed with bacteria that have been chemically coaxed into a physiological state in which they take up DNA from outside the cell. Having taken up the recombinant DNA, the bacterial cells are transferred into growth medium, where they multiply. Since the vector part of the recombinant DNA molecule contains all the DNA sequences needed for its replication and partition into the daughter cells, the recombinant DNA multiplies as the bacterial cell multiplies. If the recombinant DNA functions inside the bacterial cell, then new genetic characteristics may be expressed by the bacteria. For example, the recombinant DNA may allow the bacterial cells to produce a human protein, such as insulin or growth hormone.

Quick Check 4 In making recombinant DNA molecules that combine restriction fragments from different organisms, researchers usually prefer restriction enzymes like BamHI or HindIII that generate fragments with “sticky ends” (ends with overhangs) rather than enzymes like HpaI or SmaI (Table 12.1) that generate fragments with “blunt ends” (ends without overhangs). Can you think of a reason for this preference?

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