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Genes are the central focus of genetics, and so clearly it is desirable to be able to isolate a gene of interest (or any DNA region) from the genome and amplify it to obtain a working amount to study. DNA technology is a term that describes the collective techniques for obtaining, amplifying, and manipulating specific DNA fragments. Since the mid-1970s, the development of DNA technology has revolutionized the study of biology, opening many areas of research to molecular investigation. Genetic engineering, the application of DNA technology to specific biological, medical, or agricultural problems, is now a well-established branch of technology. Genomics is the ultimate extension of the technology to the global analysis of the nucleic acids present in a nucleus, a cell, an organism, or a group of related species (Chapter 12). How can working samples of individual DNA segments be isolated? That task initially might seem like finding a needle in a haystack. A crucial insight was that researchers could create the large samples of DNA that they needed by tricking the DNA replication machinery to replicate the DNA segment in question. Such replication could be done either within live bacterial cells (in vivo) or in a test tube (in vitro). In the in vivo approach (Figure 11-1a), the investigator begins with a sample of DNA molecules containing the gene of interest. This sample is called the donor DNA and most often it is an entire genome. Fragments of the donor DNA are inserted into nonessential “accessory” chromosomes (such as plasmids or modified bacterial viruses).

These accessory chromosomes will “carry” and amplify the gene of interest and are hence called vectors. First, the donor DNA molecules are cut up, by using enzymes called restriction endonucleases as molecular “scissors.” These enzymes are a class of DNA-binding proteins that bind to the DNA and cut the sugar–phosphate backbone of each of the two strands of the double helix at a specific sequence. They cut long chromosome-sized DNA molecules into hundreds or thousands of fragments of more manageable size. Next, each fragment is fused with a cut vector chromosome to form recombinant DNA molecules. Union with the vector DNA typically depends on short terminal single strands produced by the restriction enzymes. They bond to complementary sequences at the ends of the vector DNA. (The ends act like Velcro to join the different DNA molecules together to produce the recombinant DNA.) The recombinant DNAs are inserted into bacterial cells, and generally, only one recombinant molecule is taken up by each cell. Because the accessory chromosome is normally amplified by replication, the recombinant molecule is similarly amplified during the growth and division of the bacterial cell in which the chromosome resides. This process results in a clone of identical cells, each containing the recombinant DNA molecule, and so this technique of amplification is called DNA cloning. The next stage is finding the rare clone containing the DNA of interest. In the in vitro approach (Figure 11-1b), a specific gene of interest is amplified chemically by replication machinery extracted from special bacteria. The system “finds” the gene of interest by the complementary binding of specific short primers to the ends of that sequence. 

These primers then guide the replication process, which cycles exponentially, resulting in a large sample of copies of the gene of interest. We will see repeatedly that DNA technology depends on two basic foundations of molecular biology research: • The ability of specific proteins to recognize and bind to specific base sequences, within the DNA double helix (examples are shown in yellow in Figure 11-1). • The ability of complementary single-stranded DNA or RNA sequences to spontaneously unite to form double-stranded molecules. Examples are the binding of the sticky ends and the binding of the primers. The remainder of the chapter will explore examples of uses to which we put amplified DNA. These uses range from routine gene isolation for basic biological research to gene-based therapy of human disease. To illustrate how recombinant DNA is made, let’s consider the cloning of the gene for human insulin, a protein hormone used in the treatment of diabetes. Diabetes is a disease in which blood sugar levels are abnormally high either because the body does not produce enough insulin (type I diabetes) or because cells are unable to respond to insulin (type II diabetes).In mild forms of type I, diabetes can be treated by dietary restrictions but, for many patients, daily insulin treatments are necessary. Until about 20 years ago, cows were the major source of insulin protein. The protein was harvested from the pancreases of animals slaughtered in meat-packing plants and purified at a large scale to eliminate the majority of proteins and other contaminants in the pancreas extracts. Then, in 1982, the first recombinant human insulin came on the drug market. Human insulin could be made purer, at a lower cost, and on an industrial scale because it was produced in bacteria by recombinant DNA techniques. The recombinant insulin is a higher proportion of the proteins in the bacterial cell; hence protein purification is much easier. 

We shall follow the general steps necessary for making any recombinant DNA and apply them to insulin. Type of donor DNA The choice of DNA to be used as the donor might seem to be obvious, but there are actually three possibilities. • Genomic DNA. This DNA is obtained directly from the chromosomes of the organism under study. It is the most straightforward source of DNA. It needs to be cut up before cloning is possible. • cDNA. Complementary DNA (cDNA) is a double-stranded DNA version of an mRNA molecule. In higher eukaryotes, an mRNA is a more useful predictor of a polypeptide sequence than is a genomic sequence, because the introns have been spliced out. Researchers prefer to use cDNA rather than mRNA itself because RNAs are inherently less stable than DNA and techniques for routinely amplifying and purifying individual RNA molecules do not exist. The cDNA is made from mRNA with the use of a special enzyme called reverse transcriptase, originally isolated from retroviruses. Using an mRNA molecule as a template, reverse transcriptase synthesizes a single-stranded DNA molecule that can then be used as a template for double-stranded DNA synthesis (Figure 11-2 ).