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Recombinant DNA (rDNA) molecules areDNA molecules formed by laboratory methods ofgenetic recombination (such asmolecular cloning) that bring together genetic material from multiple sources, creatingsequences that would not otherwise be found in thegenome.
Recombinant DNA is the general name for a piece of DNA that has been created by combining two or more fragments from different sources. Recombinant DNA is possible because DNA molecules from all organisms share the same chemical structure, differing only in thenucleotide sequence. Recombinant DNA molecules are sometimes calledchimeric DNA because they can be made of material from two different species like the mythicalchimera. rDNA technology usespalindromic sequences and leads to the production ofsticky and blunt ends.
The DNA sequences used in the construction of recombinant DNA molecules can originate from anyspecies. For example, plant DNA can be joined to bacterial DNA, or human DNA can be joined with fungal DNA. In addition, DNA sequences that do not occur anywhere in nature can be created by thechemical synthesis of DNA and incorporated into recombinant DNA molecules. Using recombinant DNA technology and synthetic DNA, any DNA sequence can be created and introduced into living organisms.
Proteins that can result from the expression of recombinant DNA within living cells are termedrecombinant proteins. When recombinant DNA encoding a protein is introduced into a host organism, the recombinant protein is not necessarily produced.[1] Expression of foreign proteins requires the use of specialized expression vectors and often necessitates significant restructuring byforeign coding sequences.[2]
Recombinant DNA differs from genetic recombination in that the former results from artificial methods while the latter is a normal biological process that results in the remixing of existing DNA sequences in essentially all organisms.
Molecular cloning is the laboratory process used to produce recombinant DNA.[3][4][5][6] It is one of two most widely used methods, along withpolymerase chain reaction (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.[citation needed]
Formation of recombinant DNA requires acloning vector, a DNA molecule that replicates within a living cell. Vectors are generally derived fromplasmids orviruses, 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.[7] The DNA segments can be combined by using a variety of methods, such as restriction enzyme/ligase cloning orGibson assembly.[citation needed]
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.[6]
DNA expression requires the transfection of suitable host cells. Typically, either bacterial, yeast, insect, or mammalian cells (such ashuman embryonic kidney cells orChinese hamster ovary cells) are used as host cells.[8]
Following transplantation into the host organism, the foreign DNA contained within the recombinant DNA construct may or may not beexpressed. That is, the DNA may simply be replicated without expression, or it may betranscribed andtranslated 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 anmRNA molecule that can be used by the host'stranslational apparatus (e.g.promoter,translational initiation signal, andtranscriptional terminator).[9] 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.[10][11][12]
In most cases, organisms containing recombinant DNA have apparently normalphenotypes. 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.[13] 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 usingRT-PCR orwestern hybridization methods.[13] 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.[14] Additional phenotypes that are encountered include toxicity to the host organism induced by the recombinant gene product, especially if it isover-expressed or expressed within inappropriate cells or tissues.[citation needed]
In some cases, recombinant DNA can have deleterious effects even if it is not expressed. One mechanism by which this happens isinsertional 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.[15] 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.[citation needed]
Recombinant DNA is widely used inbiotechnology, medicine and research. Today, recombinant proteins and other products that result from the use of DNA technology are found in essentially every 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,home medicine cabinets, and even pet shops, such as those that sellGloFish and othergenetically modified animals.[citation needed]
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.[13] 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.[4]
Many additional practical applications of recombinant DNA are found in industry, food production, human and veterinary medicine, agriculture, and bioengineering.[4] Some specific examples are identified below.
Found inrennet,chymosin is the enzyme responsible for hydrolysis ofκ-casein to produce para-κ-casein andglycomacropeptide, which is the first step in formation ofcheese, and subsequentlycurd, andwhey.[16] 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) ofE. 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.[17]
Recombinant humaninsulin has almost completely replaced insulin obtained from animal sources (e.g. pigs and cattle) for the treatment oftype 1 diabetes. A variety of different recombinant insulin preparations are in widespread use.[18] Recombinant insulin (insulin aspart) is synthesized by inserting the human insulin gene intoE. coli or yeast (Saccharomyces cerevisiae), which then produces insulin for human use.[19] Insulin produced byE. coli requires furtherpost translational modifications (e.g. glycosylation) whereas yeasts are able to perform these modifications themselves by virtue of being more complex host organisms. The advantage of recombinant human insulin is after chronic use patients do not develop an immune defence against it the way animal-sourced insulin stimulates the human immune system.[20]
Human growth hormone is 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 developingCreutzfeldt–Jakob disease. Recombinant HGH eliminated this problem, and is now used therapeutically.[21] It has also been misused as a performance-enhancing drug by athletes and others.[22][23][failed verification]
Factor VIII is a blood-clotting protein that is administered to patients with the bleeding disorderhemophilia, who are unable to produce factor VIII in quantities sufficient to support normal blood coagulation.[24] 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 ofblood borne infectious diseases, for example HIV and hepatitis B.[citation needed]
Hepatitis B infection can be successfully controlled through the use of a recombinantsubunithepatitis 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 aspolio virus, cannot be grownin vitro.[25]
Recombinant antibodies (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.[26]
Each of the three widely used methods fordiagnosing HIV infection has been developed using recombinant DNA. The antibody test (ELISA orwestern blot) uses a recombinant HIV protein to test for the presence ofantibodies that the body has produced in response to an HIV infection. The DNA test looks for the presence of HIV genetic material usingreverse transcription polymerase chain reaction (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 is a recombinant variety of rice that has been engineered to express the enzymes responsible forβ-carotene biosynthesis.[14] This variety of rice holds substantial promise for reducing the incidence ofvitamin A deficiency in the world's population.[27] Golden rice is not currently in use, pending the resolution of regulatory and intellectual property issues.[28]
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 herbicideglyphosate (trade nameRoundup), and simplifies weed control by glyphosate application.[29] These crops are in common commercial use in several countries.[citation needed]
Bacillus thuringiensis is a bacterium that naturally produces a protein (Bt toxin) with insecticidal properties.[27] 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 thesetransgenic crops have not been fully resolved.[30]
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.[31] The first publications describing the successful production and intracellular replication of recombinant DNA appeared in 1972 and 1973, fromStanford andUCSF.[32][33][34][35] In 1980Paul Berg, a professor in the Biochemistry Department at Stanford and an author on one of the first papers[32] was awarded the Nobel Prize in Chemistry for his work on nucleic acids "with particular regard to recombinant DNA".Werner Arber,Hamilton Smith, andDaniel Nathans shared the 1978Nobel Prize in Physiology or Medicine for the discovery ofrestriction endonucleases which enhanced the techniques of rDNA technology.[citation needed]
Stanford University applied for a U.S. patent on recombinant DNA on November 4, 1974, listing the inventors asHerbert W. Boyer (professor at theUniversity of California, San Francisco) andStanley N. Cohen (professor atStanford University); this patent, U.S. 4,237,224A, was awarded on December 2, 1980.[36][37] The first licensed drug generated using recombinant DNA technology was human insulin, developed byGenentech and licensed byEli Lilly and Company.[38]
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 1975Asilomar 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 USNational Institutes of Health 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 ongenetically modified organisms andgenetically 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, termedhost cell protein, comes from the host expression system and poses a threat to the patient's health and the overall environment.[39][40]
