Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of

genetic recombination Genetic recombination (also known as genetic reshuffling) is the exchange of genetic material between different organisms which leads to production of offspring with combinations of traits that differ from those found in either parent. In eukaryo ...

(such as

molecular cloning Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word '' cloning'' refers to the fact that the meth ...

) that bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the

genome In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ...

. Recombinant DNA is the general name for a piece of DNA that has been created by combining at least two fragments from two different sources. Recombinant DNA is possible because DNA molecules from all organisms share the same chemical structure, and differ only in the

nucleotide Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecu ...

sequence within that identical overall structure. Recombinant DNA molecules are sometimes called chimeric DNA, because they can be made of material from two different species, like the mythical chimera. R-DNA technology uses palindromic sequences and leads to the production of sticky and blunt ends. The DNA sequences used in the construction of recombinant DNA molecules can originate from any

species In biology, a species is the basic unit of classification and a taxonomic rank of an organism, as well as a unit of biodiversity. A species is often defined as the largest group of organisms in which any two individuals of the appropriat ...

. For example, plant DNA may be joined to bacterial DNA, or human DNA may be joined with fungal DNA. In addition, DNA sequences that do not occur anywhere in nature may be created by the chemical synthesis of DNA, and incorporated into recombinant molecules. Using recombinant DNA technology and synthetic DNA, literally any DNA sequence may be created and introduced into any of a very wide range of living organisms. Proteins that can result from the expression of recombinant DNA within living cells are termed ''recombinant proteins''. When recombinant DNA encoding a protein is introduced into a host organism, the recombinant protein is not necessarily produced. Expression of foreign proteins requires the use of specialized expression vectors and often necessitates significant restructuring by foreign coding sequences. Recombinant DNA differs from genetic recombination in that the former results from artificial methods in the test tube, while the latter is a normal biological process that results in the remixing of existing DNA sequences in essentially all organisms.

DNA creation

Molecular cloning is the laboratory process used to create recombinant DNA.. Fourth edition is available online through the NCBI Bookshelf
link
/ref> Fifth edition available online through the NCBI Bookshelf
link
/ref> It is one of two most widely used methods, along with

polymerase chain reaction The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) ...

(PCR), used to direct the replication of any specific DNA sequence chosen by the experimentalist. There are two fundamental differences between the methods. One is that molecular cloning involves replication of the DNA within a living cell, while PCR replicates DNA in the test tube, free of living cells. The other difference is that cloning involves cutting and pasting DNA sequences, while PCR amplifies by copying an existing sequence. Formation of recombinant DNA requires a

cloning vector A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, ...

, a DNA molecule that replicates within a living cell. Vectors are generally derived from

plasmid A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...

s or

virus A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea. Since Dmitri Ivanovsk ...

es, and represent relatively small segments of DNA that contain necessary genetic signals for replication, as well as additional elements for convenience in inserting foreign DNA, identifying cells that contain recombinant DNA, and, where appropriate, expressing the foreign DNA. The choice of vector for molecular cloning depends on the choice of host organism, the size of the DNA to be cloned, and whether and how the foreign DNA is to be expressed. The DNA segments can be combined by using a variety of methods, such as restriction enzyme/ligase cloning or Gibson assembly. In standard cloning protocols, the cloning of any DNA fragment essentially involves seven steps: (1) Choice of host organism and cloning vector, (2) Preparation of vector DNA, (3) Preparation of DNA to be cloned, (4) Creation of recombinant DNA, (5) Introduction of recombinant DNA into the host organism, (6) Selection of organisms containing recombinant DNA, and (7) Screening for clones with desired DNA inserts and biological properties. ''These steps are described in some detail in a related article (

).''

DNA expression

Following transplantation into the host organism, the foreign DNA contained within the recombinant DNA construct may or may not be expressed. That is, the DNA may simply be replicated without expression, or it may be transcribed and

translated Translation is the communication of the meaning of a source-language text by means of an equivalent target-language text. The English language draws a terminological distinction (which does not exist in every language) between ''transla ...

and a recombinant protein is produced. Generally speaking, expression of a foreign gene requires restructuring the gene to include sequences that are required for producing an

mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein. mRNA is created during the ...

molecule that can be used by the host's translational apparatus (e.g. promoter, translational initiation signal, and transcriptional terminator). Specific changes to the host organism may be made to improve expression of the ectopic gene. In addition, changes may be needed to the coding sequences as well, to optimize translation, make the protein soluble, direct the recombinant protein to the proper cellular or extracellular location, and stabilize the protein from degradation.

Properties of organisms containing recombinant DNA

In most cases, organisms containing recombinant DNA have apparently normal

phenotype In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology (biology), morphology or physical form and structure, its Developmental biology, developmental proc ...

s. That is, their appearance, behavior and metabolism are usually unchanged, and the only way to demonstrate the presence of recombinant sequences is to examine the DNA itself, typically using a polymerase chain reaction (PCR) test. Significant exceptions exist, and are discussed below. If the rDNA sequences encode a gene that is expressed, then the presence of RNA and/or protein products of the recombinant gene can be detected, typically using

RT-PCR Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase chai ...

or western hybridization methods. Gross phenotypic changes are not the norm, unless the recombinant gene has been chosen and modified so as to generate biological activity in the host organism. Additional phenotypes that are encountered include toxicity to the host organism induced by the recombinant gene product, especially if it is over-expressed or expressed within inappropriate cells or tissues. In some cases, recombinant DNA can have deleterious effects even if it is not expressed. One mechanism by which this happens is insertional inactivation, in which the rDNA becomes inserted into a host cell's gene. In some cases, researchers use this phenomenon to " knock out" genes to determine their biological function and importance. Another mechanism by which rDNA insertion into chromosomal DNA can affect gene expression is by inappropriate activation of previously unexpressed host cell genes. This can happen, for example, when a recombinant DNA fragment containing an active promoter becomes located next to a previously silent host cell gene, or when a host cell gene that functions to restrain gene expression undergoes insertional inactivation by recombinant DNA.

Applications of recombinant DNA

Recombinant DNA is widely used in

biotechnology Biotechnology is the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services. The term ''biotechnology'' was first used ...

medicine Medicine is the science and practice of caring for a patient, managing the diagnosis, prognosis, prevention, treatment, palliation of their injury or disease, and promoting their health. Medicine encompasses a variety of health care pr ...

and

research Research is "creative and systematic work undertaken to increase the stock of knowledge". It involves the collection, organization and analysis of evidence to increase understanding of a topic, characterized by a particular attentiveness ...

. Today, recombinant proteins and other products that result from the use of DNA technology are found in essentially every western pharmacy, physician or veterinarian office, medical testing laboratory, and biological research laboratory. In addition, organisms that have been manipulated using recombinant DNA technology, as well as products derived from those organisms, have found their way into many farms,

supermarkets A supermarket is a self-service shop offering a wide variety of food, beverages and household products, organized into sections. This kind of store is larger and has a wider selection than earlier grocery stores, but is smaller and more l ...

, home medicine cabinets, and even pet shops, such as those that sell GloFish and other genetically modified animals. The most common application of recombinant DNA is in basic research, in which the technology is important to most current work in the biological and biomedical sciences. Recombinant DNA is used to identify, map and sequence genes, and to determine their function. rDNA probes are employed in analyzing gene expression within individual cells, and throughout the tissues of whole organisms. Recombinant proteins are widely used as reagents in laboratory experiments and to generate antibody probes for examining protein synthesis within cells and organisms. Many additional practical applications of recombinant DNA are found in industry, food production, human and veterinary medicine, agriculture, and bioengineering. Some specific examples are identified below. ;Recombinant chymosin Found in

rennet Rennet () is a complex set of enzymes produced in the stomachs of ruminant mammals. Chymosin, its key component, is a protease enzyme that curdles the casein in milk. In addition to chymosin, rennet contains other enzymes, such as pepsin an ...

, chymosin is an enzyme required to manufacture cheese. It was the first genetically engineered food additive used commercially. Traditionally, processors obtained chymosin from rennet, a preparation derived from the fourth stomach of milk-fed calves. Scientists engineered a non-pathogenic strain (K-12) of ''E. coli'' bacteria for large-scale laboratory production of the enzyme. This microbiologically produced recombinant enzyme, identical structurally to the calf derived enzyme, costs less and is produced in abundant quantities. Today about 60% of U.S. hard cheese is made with genetically engineered chymosin. In 1990, FDA granted chymosin " generally recognized as safe" (GRAS) status based on data showing that the enzyme was safe. ;Recombinant human

insulin Insulin (, from Latin ''insula'', 'island') is a peptide hormone produced by beta cells of the pancreatic islets encoded in humans by the ''INS'' gene. It is considered to be the main anabolic hormone of the body. It regulates the metabolism ...

: Almost completely replaced insulin obtained from animal sources (e.g. pigs and cattle) for the treatment of insulin-dependent

diabetes Diabetes, also known as diabetes mellitus, is a group of metabolic disorders characterized by a high blood sugar level ( hyperglycemia) over a prolonged period of time. Symptoms often include frequent urination, increased thirst and increased ...

. A variety of different recombinant insulin preparations are in widespread use. Recombinant insulin is synthesized by inserting the human insulin gene into '' E. coli'', or yeast (Saccharomyces cerevisiae) which then produces insulin for human use. ;Recombinant human

growth hormone Growth hormone (GH) or somatotropin, also known as human growth hormone (hGH or HGH) in its human form, is a peptide hormone that stimulates growth, cell reproduction, and cell regeneration in humans and other animals. It is thus important in ...

(HGH, somatotropin) : Administered to patients whose pituitary glands generate insufficient quantities to support normal growth and development. Before recombinant HGH became available, HGH for therapeutic use was obtained from pituitary glands of cadavers. This unsafe practice led to some patients developing Creutzfeldt–Jakob disease. Recombinant HGH eliminated this problem, and is now used therapeutically. It has also been misused as a performance-enhancing drug by athletes and others.DrugBank entry
;Recombinant blood clotting

factor VIII Factor VIII (FVIII) is an essential blood-clotting protein, also known as anti-hemophilic factor (AHF). In humans, factor VIII is encoded by the ''F8'' gene. Defects in this gene result in hemophilia A, a recessive X-linked coagulation disorder ...

: A blood-clotting protein that is administered to patients with forms of the bleeding disorder hemophilia, who are unable to produce factor VIII in quantities sufficient to support normal blood coagulation. Before the development of recombinant factor VIII, the protein was obtained by processing large quantities of human blood from multiple donors, which carried a very high risk of transmission of blood borne infectious diseases, for example HIV and hepatitis B
DrugBank entry
;Recombinant hepatitis B vaccine :

Hepatitis B Hepatitis B is an infectious disease caused by the '' Hepatitis B virus'' (HBV) that affects the liver; it is a type of viral hepatitis. It can cause both acute and chronic infection. Many people have no symptoms during an initial infection. ...

infection is controlled through the use of a recombinant hepatitis B vaccine, which contains a form of the hepatitis B virus surface antigen that is produced in yeast cells. The development of the recombinant subunit vaccine was an important and necessary development because hepatitis B virus, unlike other common viruses such as polio virus, cannot be grown

in vitro ''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called " test-tube experiments", these studies in biology a ...

Vaccine information from Hepatitis B Foundation
;Recombinant antibodies:

Recombinant antibodies Recombinant antibodies are antibody fragments produced by using recombinant antibody coding genes. They mostly consist of a heavy and light chain of the variable region of immunoglobulin. Recombinant antibodies have many advantages in both medica ...

(rAbs) are produced in vitro by the means of expression systems based on mammalian cells. Their monospecific binding to a specific epitope makes rAbs eligible not only for research purposes, but also as therapy options against certain cancer types, infections and autoimmune diseases. ;Diagnosis of infection with HIV : Each of the three widely used methods for diagnosing HIV infection has been developed using recombinant DNA. The antibody test (

ELISA The enzyme-linked immunosorbent assay (ELISA) (, ) is a commonly used analytical biochemistry assay, first described by Eva Engvall and Peter Perlmann in 1971. The assay uses a solid-phase type of enzyme immunoassay (EIA) to detect the presen ...

western blot The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detect ...

) uses a recombinant HIV protein to test for the presence of

antibodies An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of ...

that the body has produced in response to an HIV infection. The DNA test looks for the presence of HIV genetic material using

reverse transcription polymerase chain reaction Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase ch ...

(RT-PCR). Development of the RT-PCR test was made possible by the molecular cloning and sequence analysis of HIV genomes
HIV testing page from US Centers for Disease Control (CDC)
; Golden rice : A recombinant variety of rice that has been engineered to express the enzymes responsible for β-carotene biosynthesis. This variety of rice holds substantial promise for reducing the incidence of

vitamin A deficiency Vitamin A deficiency (VAD) or hypovitaminosis A is a lack of vitamin A in blood and tissues. It is common in poorer countries, especially among children and women of reproductive age, but is rarely seen in more developed countries. Nyctalopia (ni ...

in the world's population. Golden rice is not currently in use, pending the resolution of regulatory and intellectual property issues. ; Herbicide-resistant crops : Commercial varieties of important agricultural crops (including soy, maize/corn, sorghum, canola, alfalfa and cotton) have been developed that incorporate a recombinant gene that results in resistance to the herbicide glyphosate (trade name ''Roundup''), and simplifies weed control by glyphosate application. These crops are in common commercial use in several countries. ; Insect-resistant crops : ''Bacillus thuringeiensis'' is a bacterium that naturally produces a protein ( Bt toxin) with insecticidal properties. The bacterium has been applied to crops as an insect-control strategy for many years, and this practice has been widely adopted in agriculture and gardening. Recently, plants have been developed that express a recombinant form of the bacterial protein, which may effectively control some insect predators. Environmental issues associated with the use of these

transgenic A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the ...

crops have not been fully resolved.

History

The idea of recombinant DNA was first proposed by Peter Lobban, a graduate student of Prof. Dale Kaiser in the Biochemistry Department at Stanford University Medical School. The first publications describing the successful production and intracellular replication of recombinant DNA appeared in 1972 and 1973, from

Stanford Stanford University, officially Leland Stanford Junior University, is a Private university, private research university in Stanford, California. The campus occupies , among the largest in the United States, and enrolls over 17,000 students. S ...

and

UCSF The University of California, San Francisco (UCSF) is a public land-grant research university in San Francisco, California. It is part of the University of California system and is dedicated entirely to health science and life science. It con ...

. In 1980 Paul Berg, a professor in the Biochemistry Department at Stanford and an author on one of the first papers was awarded the Nobel Prize in Chemistry for his work on nucleic acids "with particular regard to recombinant DNA". Werner Arber, Hamilton Smith, and Daniel Nathans shared the 1978

Nobel Prize in Physiology or Medicine The Nobel Prize in Physiology or Medicine is awarded yearly by the Nobel Assembly at the Karolinska Institute for outstanding discoveries in physiology or medicine. The Nobel Prize is not a single prize, but five separate prizes that, accordi ...

for the discovery of

restriction endonucleases A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class ...

which enhanced the techniques of rDNA technology.

Stanford University Stanford University, officially Leland Stanford Junior University, is a private research university in Stanford, California. The campus occupies , among the largest in the United States, and enrolls over 17,000 students. Stanford is conside ...

applied for a US patent on recombinant DNA in 1974, listing the inventors as Herbert W. Boyer (professor at the

University of California, San Francisco The University of California, San Francisco (UCSF) is a public land-grant research university in San Francisco, California. It is part of the University of California system and is dedicated entirely to health science and life science. It ...

) and

Stanley N. Cohen Stanley Norman Cohen (born February 17, 1935) is an American geneticist and the Kwoh-Ting Li Professor in the Stanford University School of Medicine. Stanley Cohen and Herbert Boyer were the first scientists to transplant genes from one living ...

(professor at

); this patent was awarded in 1980. The first licensed drug generated using recombinant DNA technology was human insulin, developed by

Genentech Genentech, Inc., is an American biotechnology corporation headquartered in South San Francisco, California. It became an independent subsidiary of Roche in 2009. Genentech Research and Early Development operates as an independent center within ...

and licensed by

Eli Lilly and Company Eli Lilly and Company is an American pharmaceutical company headquartered in Indianapolis, Indiana, with offices in 18 countries. Its products are sold in approximately 125 countries. The company was founded in 1876 by, and named after, Colonel ...

Controversy

Scientists associated with the initial development of recombinant DNA methods recognized that the potential existed for organisms containing recombinant DNA to have undesirable or dangerous properties. At the 1975 Asilomar Conference on Recombinant DNA, these concerns were discussed and a voluntary moratorium on recombinant DNA research was initiated for experiments that were considered particularly risky. This moratorium was widely observed until the

National Institutes of Health The National Institutes of Health, commonly referred to as NIH (with each letter pronounced individually), is the primary agency of the United States government responsible for biomedical and public health research. It was founded in the lat ...

(USA) developed and issued formal guidelines for rDNA work. Today, recombinant DNA molecules and recombinant proteins are usually not regarded as dangerous. However, concerns remain about some organisms that express recombinant DNA, particularly when they leave the laboratory and are introduced into the environment or food chain. These concerns are discussed in the articles on

genetically modified organism A genetically modified organism (GMO) is any organism whose genetic material has been altered using genetic engineering techniques. The exact definition of a genetically modified organism and what constitutes genetic engineering varies, wit ...

s and genetically modified food controversies. Furthermore, there are concerns about the by-products in biopharmaceutical production, where recombinant DNA result in specific protein products. The major by-product, termed

host cell protein Host cell proteins (HCPs) are process-related protein impurities that are produced by the host organism during biotherapeutic manufacturing and production. During the purification process, a majority of produced HCPs are removed from the final pro ...

, comes from the host expression system and poses a threat to the patient's health and the overall environment.

References

External links

Recombinant DNA fact sheet
(from University of New Hampshire)

(Fact sheet from San Diego State University)

* ttps://archive.org/stream/dnaresearchucsf00boyerich#page/n0/mode/1up Recombinant DNA research at UCSF and commercial application at GenentechEdited transcript of 1994 interview with Herbert W. Boyer, Living history project. Oral history.
Recombinant Protein Purification Principles and Methods Handbook
*Massachusetts Institute of Technology, Oral History Program
Oral History Collection on the Recombinant DNA Controversy
MC-0100. Massachusetts Institute of Technology, Department of Distinctive Collections, Cambridge, Massachusetts {{DEFAULTSORT:Recombinant Dna American inventions Biotechnology Genetics techniques Molecular genetics Molecular biology Synthetic biology 1972 in biotechnology