Genetic Engineering

Since the discovery of the structure of DNA in the early 1950s, techniques have been developed that enable scientists not only to determine the base sequence of a particular DNA molecule but to modify that sequence by the addition or deletion of specific bases, altering in a controlled manner the message encoded by the DNA. Through the use of these techniques, it may become possible to successfully replace mutated genes in specific cells with normal genes. We end this chapter with a discussion of some of the ways in which DNA can be studied and manipulated.

Vander et al.: Human Physiology: The Mechanism of Body Function, Eighth Edition

Genetic Information and Protein Synthesis CHAPTER FIVE

Genetic Information and Protein Synthesis CHAPTER FIVE

In order to manipulate a gene, it must first be identified among the many thousands of genes in the genome, isolated in sufficient quantities to allow a determination of its base sequence, and finally inserted back into a living cell. Several methods are available for performing each of these steps.

One of the key factors in solving each of these problems is a class of bacterial enzymes called restriction nucleases, which bind to specific sequences in DNA that are four to six nucleotides long and are called restriction sites. The enzyme cuts each of the two strands of DNA at these sites. Since there are numerous restriction sites located along a large molecule of DNA and a number of restriction nucleases with different binding site specificities, the use of multiple enzymes produces a number of small DNA fragments of varying lengths, some of which may contain the complete sequence of a gene while most contain only a fragment of a gene. This reduction in the size of the DNA fragments allows various procedures to be performed that cannot be carried out on the very large molecules of intact DNA.

One application of restriction nucleases is in a procedure known as DNA fingerprinting, which can be used in an attempt to identify a particular individual

(for example, a person alleged to be involved in a crime) by analysis of blood or tissue fragments found at the scene of the crime. The DNA from these tissue samples is subjected to digestion by restriction nucle-ases, producing fragments of varying lengths. These fragments are then separated by a technique called gel electrophoresis, in which the fragments are placed at one end of a gel and subjected to an electrical current that causes the fragments to move along the gel at rates dependent on their electrical charge and size, separating the fragments into bands at different positions along the gel. Since no two individuals, with the exception of identical twins, have inherited the same combination of alleles and thus DNA sequences, different individuals produce different-sized restriction fragments. Comparing the pattern of the sample with the pattern from the tissue of a suspect can then be used to establish the probability that the two samples came from the same individual.

Restriction enzymes also provide a way to cut and paste genes between different DNA molecules. This results from the way in which restriction nucleases break the two strands of DNA. The two strands are broken at slightly different points (Figure 5-15) such that the end of one strand has a short, exposed sequence of

Donor DNA

Break points

Restriction enzymes

Host DNA-

Donor DNA fragment inserted into host DNA

Recombinant DNA

Breaks sealed by ligase

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