Using Restriction Enzymes and Plasmids to Clone a Gene

We will use the example of the insulin gene to explain how gene cloning works (figure 5.3). Insulin was the first protein to have its amino acid sequence deciphered. This is because it is relatively small (see table 4.1). It is also one that is very important medically. Figure 5.3.A shows a part of the amino acid sequence of human insulin. Because we know the genetic code if we know the sequence of amino acids of a protein, we can make a sequence of DNA, a gene, that corresponds to the sequence of amino acids in that protein. Figure 5.3.B shows the sequence of DNA corresponding to the amino acid sequence shown in figure 5.3.A. Because many amino acids can be coded for by a number of different codons, the DNA sequence shown is one of many that corresponds to the amino acid sequence. The gene coding for insulin was synthesized in this way in the laboratory. In order to have this gene transcribed at a high level in bacteria, a promoter and terminator are added to the beginning and end of the gene (figure 5.3.C). Finally, in order to clone the piece of DNA with the insulin gene on it, single-stranded ends complementary to the ends of plasmid DNA cut with a restriction enzyme are attached (figure 5.3.D). Next, scientists grow a culture of bacterial cells that contain plasmids. This plasmid DNA is purified and cut with the restriction enzyme that will produce the same complementary or "sticky" ends that were put on the piece of the insulin gene (figure 5.3.E). The engineered insulin gene is then mixed with the cut plasmid DNA. Because the cut plasmid molecules and the engineered insulin gene possess the same complementary sequences at their ends, these two DNA pieces will stick together (figure 5.3.F). Finally, the sugar-phosphate backbones of the two DNA molecules are joined together with an enzyme known as DNA ligase. The resultant piece of DNA is referred to as a "recombinant DNA molecule" because two pieces of DNA from different sources are combined.

A process similar to making a gene from a known amino acid sequence, the product of which we shall refer to as "synthetic genes," is used for other relatively small proteins of medical interest. It is also used to make the Bt toxin gene used in genetically modified plants, which will be discussed in chapter 6. However, other proteins of medical interest are far too large to use this procedure. For those proteins, a more arduous and complex cloning procedure is necessary.

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