The primary intent of recombinant DNA technology is to deliberately remove genetic material from one organism and combine it with that of a different organism. Its origins can be traced to 1970, when microbiologists first began to duplicate the clever tricks bacteria do naturally with bits of extra DNA such as plasmids, transposons, and proviruses. The discovery that bacteria can readily accept, replicate, and express foreign DNA made them powerful agents for studying the genes of other organisms in isolation. The practical applications of this work were soon realized by biotechnologists. Bacteria could be genetically engineered to mass-produce substances such as hormones, enzymes, and vaccines that have been difficult to synthesize by the usual industrial methods.
Figure 1 provides an overview of the recombinant DNA procedure. An important objective of this technique is to form genetic clones. This type of cloning involves the removal of a selected gene from an animal, plant, or microorganism (the genetic donor) followed by its propagation in a different host organism. Cloning requires that the desired donor gene first be selected, excised by restriction endonucleases, and isolated. The gene is next inserted into a vector (usually a plasmid or a virus) that will insert the DNA into a cloning host. The cloning host is usually a bacterium or a yeast that can replicate the gene and transcribe and translate it into the protein product for which it codes. Next we examine the elements of gene isolation, vectors, and cloning hosts and show how they participate in a complete recombinant DNA procedure.
Fig1. Overview of methods and applications of genetic engineering. Practical applications of genetic engineering include the development of pharmaceuticals, genetically modified organisms, and identification techniques. See process figure 3 for details of these steps used in recombinant DNA. (Animal cell): Lachina Publishing Services/McGraw Hill; (REGN-COV2): Bernard Chantal/Shutterstock; (Golden rice): JIANG HONGYAN/Shutterstock; (3D rendered illustration of DNA): vchal/Shutterstock
Technical Aspects of Recombinant DNA and Gene Cloning
The first hurdles in cloning a target gene are to locate its exact site on the genetic donor’s chromosome and to iso late it. Some of the first isolated genes came from viruses, which have extremely small and manageable genomes, and later genes were isolated from bacteria and yeasts. One of the greatest benefits of the Human Genome Project is that the exact sequence and location of each gene in the human genome is known, greatly simplifying its initial isolation. Beyond this first step, there are common strategies for obtaining isolated genes, including:
1. Synthesizing a cDNA from a mRNA transcript.
2. Isolating a gene from a larger segment of DNA using PCR.
3. Synthesizing a gene using purely chemical means. Laboratory equipment exists that can synthesize a strand of DNA containing any combination of nucleotides, even sequences that don’t exist in nature.
Although the initial phases of gene isolation and cloning can be labor-intensive, a fortunate outcome is that, once isolated, genes can be maintained in a cloning host and vector just like a microbial pure culture. Genomic libraries, which contain fragments of DNA representing all the genes in a particular species, have been created for thousands of organisms and viruses.
Characteristics of Cloning Vectors
A good recombinant vector has two indispensable qualities: It must be capable of carrying a significant piece of the donor DNA, and it must be readily accepted by the cloning host. Plasmids are excellent vectors because they are small, well characterized, and easy to manipulate, and they can be transferred into appropriate host cells through transformation. Bacteriophages also serve well because they have the natural ability to inject DNA into bacterial hosts through transduction.
Today, thousands of unique cloning vectors are available commercially. Although every vector has characteristics that make it ideal for a specific project, all vectors can be thought of as having three important attributes (figure2):
1. An origin of replication (ORI) is needed somewhere on the vector so that it will be replicated by the DNA polymerase of the cloning host.
2. The vector must accept DNA of the desired size. Early plasmids were limited to an insert size of less than 10 kb of DNA, far too small for most eukaryotic genes, with their sizable introns. Vectors called cosmids can hold 45 kb, whereas complex bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) can hold as much as 300 kb and 1,000 kb, respectively.
3. Vectors typically contain a gene that confers drug resistance to their cloning host. In this way, cells can be inoculated into drug-containing media, and only those cells that harbor a plasmid will be selected for growth.
Fig2. Partial map of the pUC18 plasmid. Each base pair is numbered, beginning with 0 at the top of the plasmid and moving clockwise. Sites cleaved by various restriction endonucleases are indicated. The multiple cloning site (MCS) is an engineered site designed so that a piece of foreign DNA may be easily inserted. Other important components are genes for ampicillin resistance (AmpR) and the origin of replication (ori). Many plasmids in use today are derivatives of pUC18. Barry Chess/McGraw Hill
Characteristics of Cloning Hosts
The best cloning hosts possess several key characteristics (table 1). The traditional cloning host—and the one still used in basic experiments—is Escherichia coli. Because this bacterium was the original recombinant host, the protocols using it are well established, relatively easy, and reliable. Hundreds of specialized cloning vectors have been developed for it. The main disadvantages with this species are that the splicing of mRNA and the modification of proteins that would normally occur in the eukaryotic endoplasmic reticulum and Golgi apparatus are unavailable in this prokaryotic cloning host. One alternative host for certain industrial processes and research is the eukaryotic yeast Saccharomyces cerevisiae, which would be able to process and modify eukaryotic genes and products.
Table1. Desirable Features in a Microbial Cloning Host
Certain techniques may also employ animal cell culture, and even live animals and plants, to serve as cloning hosts. Although a number of recombinant proteins are produced by transformed bacterial hosts, many of the enzymes, hormones, and antibodies used in drug therapy are currently being manufactured with mammalian cell cultures as the cloning and ex pression hosts. One of the primary advantages to this alternative procedure is that these cell cultures can modify the proteins (adding carbohydrates, for example) so that they are biologically more active. Some forms of reproductive hormones, human growth hormone, and interferon are products of cell culture. In our coverage, we present the recombinant process as it is per formed in bacteria and yeasts.
Construction of a Recombinant, Insertion into a Cloning Host, and Genetic Expression
This section relates the main steps in recombinant DNA technology needed to produce a protein. As an example, we will use a drug called recombinant erythropoietin (EPO), but it should be remembered that the steps are essentially the same regardless of the protein being produced. Erythropoietin stimulates the production of red blood cells by the bone marrow and is normally produced by the kidneys. People with kidney disease may produce low levels of EPO, leading to anemia. Injections of recombinant EPO increase the number of red blood cells circulating in the body. The erythropoietin gene isolated from human cells is approximately 500 bp and codes for a protein of 165 amino acids. It was originally isolated and identified from processed mRNA transcripts that are free of introns.
The first step in cloning the gene is to prepare it for splicing into a plasmid (process figure 3). This can be accomplished by digesting both the gene and the plasmid with the same restriction enzyme, resulting in complementary sticky ends on both the vector and insert DNA. When the gene and plasmid are placed together, their free ends base-pair, and ligase produces a final covalent bonds. The resultant gene and plasmid combination is called a recombinant.
Process Fig3. An overview of recombinant DNA and gene cloning.
Following this procedure, the recombinant is introduced by transformation into the cloning host, a special laboratory strain of E. coli that lacks any extra plasmids that could complicate the ex pression of the gene. Because the recombinant plasmid enters only some of the cloning host cells, it is necessary to locate these recombinant clones. Cultures are plated out on medium containing an antibiotic such as ampicillin or tetracycline, and only those clones that carry the plasmid with resistance to the antibiotic can survive and form colonies. These recombinant colonies are selected from the plates and cultured. Once the gene has been successfully cloned, this step does not have to be repeated; the recombinant strain can be maintained in culture indefinitely.
Many proteins, including erythropoietin, require posttranslational modification that can only take place within a eukaryotic cell (modification of the protein in the Golgi apparatus, for example). In such a case, the isolated gene can be easily moved to a second vector and used to transform a eukaryotic cloning host, typically cultured mammalian cells. And whether the cloning host is prokaryotic or eukaryotic, the final steps are the same; the gene is transcribed and translated by the host, and the protein is exported into the growth medium, where it is collected and purified. The scale of this procedure can range from test tube size to gigantic industrial tanks that can manufacture thousands of gallons of product (figure 4). The basic process we have presented here can be harnessed to mass produce a variety of protein products, including hormones, enzymes, vaccines, and agricultural products (see table 2).
Fig4. Recombinant protein production. Once a recombinant molecule has been created and inserted into a host cell, the host can be grown in large quantities, producing enormous quantities of the protein of interest. These bioreactors contain recombinant bacteria used in the production of pharmaceutical products. Each tank is three stories tall and contains several thousand liters of growth media. Maximilian/Prisma by Dukas Presseagentur GmbH/Alamy Stock Photo
Table2. Examples of Products Made Using Recombinant DNA Technology
Protein Products of Recombinant DNA Technology
Recombinant DNA technology is used by pharmaceutical companies to manufacture medications that are difficult to procure by other means. Diseases such as diabetes and dwarfism, caused by the lack of an essential hormone, are treated by replacing the missing hormone. Insulin of animal origin was once the only form available to treat diabetes, even though such animal products can cause allergic reactions in sensitive individuals. In contrast, pituitary dwarf ism cannot be treated with animal growth hormones, so originally the only source of human growth hormone (HGH) was the pituitaries of cadavers. At one time, not enough HGH was available to treat the thousands of children in need.
Another serious problem with using natural human products is the potential for infection. For example, infectious agents such as the prion responsible for Creutzfeldt-Jakob disease can be transmitted in this manner. Similarly, clotting fac tor VIII, a protein needed for blood to clot properly, is missing in persons suffering from hemophilia A. In the early 1980s a large percentage of the patients receiving plasma-derived factor VIII contracted HIV infections as a result of being exposed to the virus through the donated plasma.
Recombinant technology changed the outcome of these and many other conditions by enabling large-scale manufacture of hormones and enzymes derived from human genes. Recombinant human insulin was the first rDNA hormone to be marketed for diabetics, followed by recombinant HGH for children with dwarfism and Turner syndrome. HGH is also used to prevent the wasting syndrome that occurs in AIDS and cancer patients. In all of these applications, recombinant DNA technology has led to both a safer product and one that can be manufactured in mass quantities. Other protein-based hormones, enzymes, and vaccines produced through recombinant DNA are summarized in table 1.
While proteins are the traditional end product in genetic engineering, nucleic acids themselves also have a number of applications. The Moderna and Pfizer vaccines used to protect against SARS-CoV-2 infection both rely on engineered messenger RNA, and similar mRNA vaccines are in development for other diseases. Additional examples of recombinant RNA and DNA molecules used as gene therapy.
