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Modern Genetic Analysis Chapter 10. Recombinant DNA Technology
Introduction

The goal of genetics is to study the structure and function of genes and genomes. Since Mendel's time, genes have been identified by observing standard phenotypic ratios in controlled crosses. Clues about gene function first came from correlating specific mutations with enzyme and other protein deficiencies. The correlation of mutant sites within a gene with amino acid substitutions in the appropriate protein led to a better understanding of gene structure and function. To these ideas were added discoveries about the nature of DNA and the genetic code, leading to a fairly comprehensive understanding of the basic nature of the gene. However, all were indirect inferences about genes; no gene had ever been isolated and its DNA sequence examined directly. Indeed, it seemed impossible to isolate an individual gene from the genome.

Although it is relatively easy to isolate DNA from living tissue, in a test tube DNA looks like a glob of mucus. How could it be possible to isolate a single gene from this tangled mass of DNA threads? Recombinant DNA technology provides the techniques for doing just that, and today individual genes and other parts of genomes are isolated routinely.

Why is gene isolation so important? First, isolation of a gene enables the determination of its nucleotide sequence. From this information, the internal landmarks of the gene can be determinedfor example, intron number and position. A comparison of DNA sequences between genes also can lead to insights in gene evolution. Converting the DNA sequence of a gene into amino acid sequence by using the genetic code leads to a direct determination of the structure of the protein product of a gene, and, from this knowledge, the function of the gene can often be inferred. Function can also be studied by direct modification of part of the gene's DNA sequence followed by the reintroduction of the gene into the genome. Furthermore, a gene can be moved from one organism to another. An organism containing a foreign gene is called transgenic. Transgenic organisms can be used either for basic research or for specialized commercial applications. One application has been to make valuable human gene products such as insulin in transgenic bacteria carrying the appropriate human gene. From this brief overview, we see that gene isolation has become an indispensable tool of modern genetic analysis.

What are some examples of interesting genes that could be isolated? The answer depends very much on which biological process is being studied. Let's look at a few cases. A fungal geneticist studying the cellular pathway for synthesizing tryptophan would be interested in the genes that, when mutated, confer an auxotrophic requirement for tryptophan, because each gene would represent a step in the synthetic pathway (see Chapter 3). These genes can be identified through mutation, segregation, and mapping analysis. They would be named trp1, trp2, trp3, and so forth. This geneticist would be very interested in isolating and characterizing one or more of these genes. Likewise, human genes that have mutant alleles conferring some type of functional disorder are interesting for medical and biological reasons. We have seen that these genes are identified by pedigree analysis. Two examples covered in Chapters 3 and 4 are the recessive autosomal conditions albinism and alkaptonuria. In these cases, the general nature of the defect has been understood for some time (both are enzyme defects), but it would be very useful to isolate the genes themselves. Other human genes are known from pedigree analysis, but no biochemical function is known for them. Isolating such genes would be particularly useful because the characterization of gene structure might lead to a determination of gene function and the nature of the disease. A good example is cystic fibrosis, a disease known from pedigree analysis to be caused by an autosomal recessive allele of a gene for which no function was known until the gene was isolated and sequenced. Cases such as these could be raised in all the organisms used in genetic research.

In our consideration of gene isolation, we shall first examine the nature of recombinant DNA and the principle whereby recombinant DNA technology can be used to isolate a gene. Next, we shall examine the methods for isolating specific genes such as those just discussed.

© 1999 by W. H. Freeman and Company. All rights reserved.