Recombinant DNA

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Construction of recombinant DNA, in which a foreign DNA fragment is inserted into a plasmid vector. In this example, the gene indicated by the white color is inactivated upon insertion of the foreign DNA fragment. Recombinant formation of plasmids.svg
Construction of recombinant DNA, in which a foreign DNA fragment is inserted into a plasmid vector. In this example, the gene indicated by the white color is inactivated upon insertion of the foreign DNA fragment.

Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome.

Contents

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 the nucleotide sequence. Recombinant DNA molecules are sometimes called chimeric DNA because they can be made of material from two different species like the mythical chimera. rDNA 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. 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 the chemical 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 termed recombinant 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 by foreign 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.

Production

Gene cloning.svg

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 with polymerase 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.

Formation of recombinant DNA requires a cloning vector, a DNA molecule that replicates within a living cell. Vectors are generally derived from plasmids or viruses, 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 or Gibson 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] These steps are described in some detail in a related article (molecular cloning).

DNA expression

DNA expression requires the transfection of suitable host cells. Typically, either bacterial, yeast, insect, or mammalian cells (such as Human Embryonic Kidney cells or CHO 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 be expressed. That is, the DNA may simply be replicated without expression, or it may be transcribed and translated 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 molecule that can be used by the host's translational apparatus (e.g. promoter, translational initiation signal, and transcriptional 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]

Properties of organisms containing recombinant DNA

In most cases, organisms containing recombinant DNA have apparently normal phenotypes. 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 using RT-PCR or western 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 is over-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 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. [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 ]

Applications of recombinant DNA

Recombinant DNA is widely used in biotechnology, medicine and research. 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, 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. [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.

Recombinant chymosin

Found in rennet, chymosin is the enzyme responsible for hydrolysis of κ-casein to produce para-κ-casein and glycomacropeptide, which is the first step in formation of cheese, and subsequently curd, and whey. [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) 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. [17]

Recombinant human insulin

Recombinant human insulin has almost completely replaced insulin obtained from animal sources (e.g. pigs and cattle) for the treatment of type 1 diabetes. A variety of different recombinant insulin preparations are in widespread use. [18] Recombinant insulin is synthesized by inserting the human insulin gene into E. coli , or yeast (Saccharomyces cerevisiae) [19] which then produces insulin for human use. [20] Insulin produced by E. coli requires further post 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 don't develop an immune defence against it the way animal sourced insulin stimulates the human immune system. [21]

Recombinant human growth hormone (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. [22] It has also been misused as a performance-enhancing drug by athletes and others. [23] [24]

Recombinant blood clotting factor VIII

It is the recombinant form of factor VIII, a blood-clotting protein that is administered to patients with the bleeding disorder hemophilia, who are unable to produce factor VIII in quantities sufficient to support normal blood coagulation. [25] 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.

Recombinant hepatitis B vaccine

Hepatitis B infection can be successfully controlled through the use of a recombinant subunit 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. [26]

Recombinant antibodies

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. [27]

Diagnosis of HIV infection

Each of the three widely used methods for diagnosing HIV infection has been developed using recombinant DNA. The antibody test (ELISA or western blot) uses a recombinant HIV protein to test for the presence of antibodies 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 (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

Golden rice is a recombinant variety of rice that has been engineered to express the enzymes responsible for β-carotene biosynthesis. [14] This variety of rice holds substantial promise for reducing the incidence of vitamin A deficiency in the world's population. [28] Golden rice is not currently in use, pending the resolution of regulatory and intellectual property issues. [29]

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. [30] These crops are in common commercial use in several countries.

Insect-resistant crops

Bacillus thuringiensis is a bacterium that naturally produces a protein (Bt toxin) with insecticidal properties. [28] 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 crops have not been fully resolved. [31]

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. [32] The first publications describing the successful production and intracellular replication of recombinant DNA appeared in 1972 and 1973, from Stanford and UCSF. [33] [34] [35] [36] In 1980 Paul Berg, a professor in the Biochemistry Department at Stanford and an author on one of the first papers [33] 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 for the discovery of restriction 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 as Herbert W. Boyer (professor at the University of California, San Francisco) and Stanley N. Cohen (professor at Stanford University); this patent, U.S. 4,237,224A, was awarded on December 2, 1980. [37] [38] The first licensed drug generated using recombinant DNA technology was human insulin, developed by Genentech and licensed by Eli Lilly and Company. [39]

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 (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 organisms 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, comes from the host expression system and poses a threat to the patient's health and the overall environment. [40] [41]

See also

Related Research Articles

<span class="mw-page-title-main">Complementary DNA</span> DNA reverse transcribed from RNA

In genetics, complementary DNA (cDNA) is DNA that was reverse transcribed from an RNA. cDNA exists in both single-stranded and double-stranded forms and in both natural and engineered forms.

<span class="mw-page-title-main">Genetic engineering</span> Manipulation of an organisms genome

Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species boundaries to produce improved or novel organisms. New DNA is obtained by either isolating and copying the genetic material of interest using recombinant DNA methods or by artificially synthesising the DNA. A construct is usually created and used to insert this DNA into the host organism. The first recombinant DNA molecule was made by Paul Berg in 1972 by combining DNA from the monkey virus SV40 with the lambda virus. As well as inserting genes, the process can be used to remove, or "knock out", genes. The new DNA can be inserted randomly, or targeted to a specific part of the genome.

Molecular biology is a branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions.

<span class="mw-page-title-main">Plasmid</span> Small DNA molecule within a cell

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; however, plasmids are sometimes present in archaea and eukaryotic organisms. In nature, plasmids often carry genes that benefit the survival of the organism and confer selective advantage such as antibiotic resistance. While chromosomes are large and contain all the essential genetic information for living under normal conditions, plasmids are usually very small and contain only additional genes that may be useful in certain situations or conditions. Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms. In the laboratory, plasmids may be introduced into a cell via transformation. Synthetic plasmids are available for procurement over the internet.

<span class="mw-page-title-main">DNA vaccine</span> Vaccine containing DNA

A DNA vaccine is a type of vaccine that transfects a specific antigen-coding DNA sequence into the cells of an organism as a mechanism to induce an immune response.

<span class="mw-page-title-main">Protein production</span>

Protein production is the biotechnological process of generating a specific protein. It is typically achieved by the manipulation of gene expression in an organism such that it expresses large amounts of a recombinant gene. This includes the transcription of the recombinant DNA to messenger RNA (mRNA), the translation of mRNA into polypeptide chains, which are ultimately folded into functional proteins and may be targeted to specific subcellular or extracellular locations.

<span class="mw-page-title-main">Cloning vector</span> Small piece of maintainable DNA

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, or it may be the plasmid of a bacterium. The vector contains features that allow for the convenient insertion of a DNA fragment into the vector or its removal from the vector, for example through the presence of restriction sites. The vector and the foreign DNA may be treated with a restriction enzyme that cuts the DNA, and DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and vector DNA and foreign DNA with compatible ends can then be joined by molecular ligation. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.

<span class="mw-page-title-main">Molecular genetics</span> Scientific study of genes at the molecular level

Molecular genetics is a branch of biology that addresses how differences in the structures or expression of DNA molecules manifests as variation among organisms. Molecular genetics often applies an "investigative approach" to determine the structure and/or function of genes in an organism's genome using genetic screens. 

<span class="mw-page-title-main">Expression vector</span> Virus or plasmid designed for gene expression in cells

An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology for the production of proteins.

<span class="mw-page-title-main">Library (biology)</span>

In molecular biology, a library is a collection of DNA fragments that is stored and propagated in a population of micro-organisms through the process of molecular cloning. There are different types of DNA libraries, including cDNA libraries, genomic libraries and randomized mutant libraries. DNA library technology is a mainstay of current molecular biology, genetic engineering, and protein engineering, and the applications of these libraries depend on the source of the original DNA fragments. There are differences in the cloning vectors and techniques used in library preparation, but in general each DNA fragment is uniquely inserted into a cloning vector and the pool of recombinant DNA molecules is then transferred into a population of bacteria or yeast such that each organism contains on average one construct. As the population of organisms is grown in culture, the DNA molecules contained within them are copied and propagated.

A DNA construct is an artificially-designed segment of DNA borne on a vector that can be used to incorporate genetic material into a target tissue or cell. A DNA construct contains a DNA insert, called a transgene, delivered via a transformation vector which allows the insert sequence to be replicated and/or expressed in the target cell. This gene can be cloned from a naturally occurring gene, or synthetically constructed. The vector can be delivered using physical, chemical or viral methods. Typically, the vectors used in DNA constructs contain an origin of replication, a multiple cloning site, and a selectable marker. Certain vectors can carry additional regulatory elements based on the expression system involved.

<span class="mw-page-title-main">DNA shuffling</span>

DNA shuffling, also known as molecular breeding, is an in vitro random recombination method to generate mutant genes for directed evolution and to enable a rapid increase in DNA library size. Three procedures for accomplishing DNA shuffling are molecular breeding which relies on homologous recombination or the similarity of the DNA sequences, restriction enzymes which rely on common restriction sites, and nonhomologous random recombination which requires the use of hairpins. In all of these techniques, the parent genes are fragmented and then recombined.

<span class="mw-page-title-main">Biotechnology in pharmaceutical manufacturing</span>

Biotechnology is the use of living organisms to develop useful products. Biotechnology is often used in pharmaceutical manufacturing. Notable examples include the use of bacteria to produce things such as insulin or human growth hormone. Other examples include the use of transgenic pigs for the creation of hemoglobin in use of humans.

Biomolecular engineering is the application of engineering principles and practices to the purposeful manipulation of molecules of biological origin. Biomolecular engineers integrate knowledge of biological processes with the core knowledge of chemical engineering in order to focus on molecular level solutions to issues and problems in the life sciences related to the environment, agriculture, energy, industry, food production, biotechnology and medicine.

<span class="mw-page-title-main">Functional cloning</span>

Functional cloning is a molecular cloning technique that relies on prior knowledge of the encoded protein’s sequence or function for gene identification. In this assay, a genomic or cDNA library is screened to identify the genetic sequence of a protein of interest. Expression cDNA libraries may be screened with antibodies specific for the protein of interest or may rely on selection via the protein function. Historically, the amino acid sequence of a protein was used to prepare degenerate oligonucleotides which were then probed against the library to identify the gene encoding the protein of interest. Once candidate clones carrying the gene of interest are identified, they are sequenced and their identity is confirmed. This method of cloning allows researchers to screen entire genomes without prior knowledge of the location of the gene or the genetic sequence.

<span class="mw-page-title-main">Genetically modified bacteria</span> First organisms to be modified in the laboratory

Genetically modified bacteria were the first organisms to be modified in the laboratory, due to their simple genetics. These organisms are now used for several purposes, and are particularly important in producing large amounts of pure human proteins for use in medicine.

Heterologous expression refers to the expression of a gene or part of a gene in a host organism that does not naturally have the gene or gene fragment in question. Insertion of the gene in the heterologous host is performed by recombinant DNA technology. The purpose of heterologous expression is often to determine the effects of mutations and differential interactions on protein function. It provides an easy path to efficiently express and experiment with combinations of genes and mutants that do not naturally occur.

<span class="mw-page-title-main">Molecular cloning</span> Set of methods in molecular biology

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 method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.

<span class="mw-page-title-main">History of genetic engineering</span>

Genetic engineering is the science of manipulating genetic material of an organism. The first artificial genetic modification accomplished using biotechnology was transgenesis, the process of transferring genes from one organism to another, first accomplished by Herbert Boyer and Stanley Cohen in 1973. It was the result of a series of advancements in techniques that allowed the direct modification of the genome. Important advances included the discovery of restriction enzymes and DNA ligases, the ability to design plasmids and technologies like polymerase chain reaction and sequencing. Transformation of the DNA into a host organism was accomplished with the invention of biolistics, Agrobacterium-mediated recombination and microinjection. The first genetically modified animal was a mouse created in 1974 by Rudolf Jaenisch. In 1976 the technology was commercialised, with the advent of genetically modified bacteria that produced somatostatin, followed by insulin in 1978. In 1983 an antibiotic resistant gene was inserted into tobacco, leading to the first genetically engineered plant. Advances followed that allowed scientists to manipulate and add genes to a variety of different organisms and induce a range of different effects. Plants were first commercialized with virus resistant tobacco released in China in 1992. The first genetically modified food was the Flavr Savr tomato marketed in 1994. By 2010, 29 countries had planted commercialized biotech crops. In 2000 a paper published in Science introduced golden rice, the first food developed with increased nutrient value.

<span class="mw-page-title-main">Genetic engineering techniques</span> Methods used to change the DNA of organisms

Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.

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