Recombinant DNA is genetic material created by artificially introducing DNA from one organism, termed the donor, into the DNA of a different organism, known as the vector. Then, as it multiplies, the vector organism not only makes many copies of the recombinant DNA but also expresses the inserted DNA. An example of such expression is the production of a protein encoded by the inserted DNA. The collection of techniques that creates and analyzes the DNA molecules that contain the recombined DNA from two unrelated organisms, along with the substances produced by the merging of the two DNAs, is called recombinant DNA technology.
The organism that receives the inserted DNA molecules is typically a bacterium or virus whose DNA is capable of accepting another DNA molecule (called a vector DNA). The donor DNA molecule is from an organism with genes or gene products of interest, which could be anything from a bacterium to a human. The new combination of these two DNA molecules allows for the creation of many copies of a specific DNA through the multiplication process known as replication or cloning. Such DNA copies can then be studied in detail, used to produce valuable proteins and therapies, used for gene therapy, or used for other applications such as the making of biofuels and the creation of genetically modified organisms (GMOs).
Working at Stanford University, biochemist Paul Berg (1926–) devised an experiment that in 1971 created the first recombinant DNA (an experiment that later sparked an unforeseen controversy). Berg successfully merged a group of three genes from the bacterium Escherichia coli (E. coli) into the DNA of the SV40 virus, found in monkeys and humans. Those bacterial genes are needed to break down the sugar galactose for use by a cell, a process known as metabolism. This experimental gene splicing created the first recombinant DNA, the recognition of which earned Berg a share of the 1980 Nobel Prize in Chemistry.
Because the SV40 virus is an oncovirus (that is, a virus that causes cells to become cancerous), safety and ethical concerns about the potential risks of oncogenic viruses brought recombinant DNA experimentation to an early end, albeit temporarily. Following a 1975 meeting at Asilomar in California, participants agreed to a set of safety standards for recombinant DNA work, including the use of disabled bacteria unable to survive outside the laboratory. This conference helped satisfy the public about the safety of recombinant DNA research and led to a rapid expansion of the use of these powerful technologies. In 1973, Stanley Cohen (1936–), Herbert Boyer (1935–), and their respective Stanford and University of California, San Francisco, colleagues Annie C. Y. Chang and Robert B. Helling (1937–2006) first reported the successful cloning of DNA using a bacterial plasmid, a circular ring of DNA. Together, these discoveries ignited the field of genetic engineering and ushered in the era of recombinant DNA technology.
The basic technique of recombinant DNA involves breaking up (cleaving) a vector DNA with a restriction enzyme, a sort of molecular scissors that cut DNA at specific sites. This cleavage is known as digestion and the products of digestion, in molecular biology, are called digests. A DNA molecule from the organism of interest is also digested, in a separate tube, with the same restriction enzyme. The two DNAs are then mixed together and joined, this time using an enzyme called DNA ligase, to make an intact, double-stranded DNA molecule. This construct is then put into E. coli cells, where the resulting DNA is copied billions of times. This novel DNA molecule is then isolated from the E. coli cells and analyzed to make sure that the correct construct was produced. The nucleotide order of the DNA can then be determined (sequenced), and the DNA can be used to generate protein from E. coli or another host, or for many other purposes. Besides E. coli, recombinant cell factories have broadened to include other types of bacteria, yeasts, insect cells, and, notably, mammalian cells whose metabolic and protein-processing pathways are similar to those in human cells.
There are many variations on this basic method of producing recombinant DNA molecules. For example, sometimes researchers are interested in isolating a whole collection of DNAs from an organism. In this case, they digest the whole genome with restriction enzyme, join many DNA fragments into many different vector molecules, and then transform those molecules into E. coli. The different E. coli cells that contain different DNA molecules are then pooled, resulting in a “library” of E. coli cells that contain, collectively, all of the genes present in the original organism.
Another variation is to make a library of all expressed genes (genes that are used to make proteins) from an organism or tissue. In this case, RNA is isolated. The isolated RNA is converted to DNA using the enzyme called reverse transcriptase. The resulting complementary DNA copy, commonly abbreviated as cDNA, is then joined to vector molecules and put into E. coli. This collection of recombinant cDNAs, which makes up a cDNA library, allows researchers to study the expressed genes in an organism, independently of nonexpressed DNA.
Recombinant DNA technology has been used for many purposes. The Human Genome Project relied on recombinant DNA technology and amplification techniques such as the polymerase chain reaction (PCR) to generate libraries of genomic DNA molecules. Proteins for the treatment or diagnosis of disease have been produced using recombinant DNA techniques. Within a year of its first recombinant product, the human hormone somatostatin (1977), the first biotechnology company, Genentech, announced their development of a recombinant human insulin, which in 1982 became the first recombinant drug product approved by the Food and Drug Administration. Since these developments, other high-purity, cell-free products have been manufactured. In addition, a number of crops have been modified using these methods.
By 2017, about 500 products approved for use in the treatment of diseases or as vaccines had been produced using recombinant DNA techniques, with well over 1,000 in development. Among the recombinant DNA technology products are hepatitis B vaccine, human growth hormone, clotting factors for treating hemophilia, tissue plasminogen activator (TPA, known as a “clot-busting” drug used to treat some types of stroke), the antiviral drug interferon, enzymes, and many other drugs, including anticancer drugs. In addition, a number of diagnostic tests for diseases, including tests for hepatitis and human immunodeficiency virus (HIV; the virus that causes AIDS), have been produced using recombinant DNA technology.
Gene therapy, which attempts to correct a missing or defective gene through some type of genetic manipulation, is another area of applied genetics that requires recombinant DNA techniques. In this case, the recombinant DNA molecules themselves are used for therapy. A prominent example is CRISPR, an acronym for clustered regularly interspaced short palindromic repeats (a palindrome is a sequence that reads the same backward as forward). CRISPR uses methods pioneered by recombinant DNA technology to edit the genome by removing, replacing, or adding to parts of the DNA sequence in living eukaryotic cells. Gene therapies such as CRISPR are being developed and attempted for a number of inherited human diseases, especially diseases due to a single gene defect. Examples include certain types of anemia, such as sickle cell anemia and beta-thalassemia.
Recombinant DNA technology has also been used to produce genetically modified foods, microbes, crops, and animals. These include tomatoes that can be vine-ripened before shipping and rice with improved nutritional qualities. Genetically modified foods have generated controversy, and debate is ongoing about the benefits and risks of developing crops using recombinant DNA technology. Concerns include the emergence of super-resistance following the exposure to genetically modified crops or organisms that were originally designed for disease-resistance, improved yield, and other hardiness traits.
Another important application of recombinant biotechnology is the development of biofuels, such as bioethanol. Recombinant DNA can be used to produce purified enzymes that can convert plant carbohydrates such as cellulose (which is typically difficult to break down and ferment) into alcohol, a clean, renewable, and economical source of energy. Bioethanols used in the gasoline that fuels the combustion engines of motor vehicles produce cleaner emissions than those of fossil fuels.
Since the mid-1970s, recombinant DNA techniques have been widely applied in research laboratories and in pharmaceutical and agricultural companies. It is likely that this rapidly evolving area of genetics will continue to play an important part in biological research into the foreseeable future, driven in part by growing interest in biological therapies, vaccine development, and high-purity medicinal products free of potential contaminants.
replication
duplication of DNA
metabolism
chemical reactions within a cell
plasmid
a small ring of DNA found in many bacteria
restriction enzyme
an enzyme that cuts DNA at a particular sequence
ligase
enzyme that repairs breaks in DNA
genome
the total genetic material in a cell or organism
amplification
multiplication
PCR
polymerase chain reaction, a technique used to produce many copies of DNA
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Matilde Parente