Recombinant DNA molecules are
DNADeoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
sequences that result from the use of laboratory methods (
molecular cloningMolecular cloning refers to a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms...
) to bring together genetic material from multiple sources, creating
sequencesThe sequence or primary structure of a nucleic acid is the composition of atoms that make up the nucleic acid and the chemical bonds that bond those atoms. Because nucleic acids, such as DNA and RNA, are unbranched polymers, this specification is equivalent to specifying the sequence of...
that would not otherwise be found in biological organisms. Recombinant DNA is possible because DNA molecules from all organisms share the same chemical structure; they differ only in the sequence of nucleotides within that identical overall structure. Consequently, when DNA from a foreign source is linked to host sequences that can drive
DNA replicationDNA replication is a biological process that occurs in all living organisms and copies their DNA; it is the basis for biological inheritance. The process starts with one double-stranded DNA molecule and produces two identical copies of the molecule...
and then introduced into a host organism, the foreign DNA is replicated along with the host DNA.
Introduction
Recombinant DNA molecules are sometimes called
chimeric DNA, because they are usually made of material from two different species, like the mythological
chimeraThe Chimera or Chimaera was, according to Greek mythology, a monstrous fire-breathing female creature of Lycia in Asia Minor, composed of the parts of multiple animals: upon the body of a lioness with a tail that ended in a snake's head, the head of a goat arose on her back at the center of her...
.
The DNA sequences used in the construction of recombinant DNA molecules can originate from any
speciesIn biology, a species is one of the basic units of biological classification and a taxonomic rank. A species is often defined as a group of organisms capable of interbreeding and producing fertile offspring. While in many cases this definition is adequate, more precise or differing measures are...
. 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 DNAOligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleic acids with defined chemical structure . The technique is extremely useful in current laboratory practice because it provides a rapid and inexpensive access to custom-made oligonucleotides of the desired...
, 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 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 will not necessarily be produced. Expression of foreign proteins requires the use of specialized
expression vectorAn expression vector, otherwise known as an expression construct, is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular-transcription and translation...
s and often necessitates significant restructuring of the foreign coding sequence.
It is important to note that recombinant DNA differs from
genetic recombinationGenetic recombination is a process by which a molecule of nucleic acid is broken and then joined to a different one. Recombination can occur between similar molecules of DNA, as in homologous recombination, or dissimilar molecules, as in non-homologous end joining. Recombination is a common method...
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.
Creating recombinant DNA
Molecular cloning is the laboratory process used to create recombinant DNA. It is one of two widely-used methods (along with
polymerase chain reactionThe polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
, abbr. PCR) used to direct the replication of any specific DNA sequence chosen by the experimentalist. The fundamental difference between the two methods 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.
Formation of recombinant DNA requires a
cloning vectorIn molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, viruses, cosmids, and artificial chromosomes...
, a DNA molecule that will replicate within a living cell. Vectors are generally derived from
plasmidIn microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
s or
virusA virus is a small infectious agent that can replicate only inside the living cells of organisms. Viruses infect all types of organisms, from animals and plants to bacteria and archaea...
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 assemblyGibson assembly is a DNA assembly method which allows for the joining of multiple DNA fragments in a single, isothermal reaction. It was invented in 2009 by the Daniel Gibson while he was at the J...
.
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, (7) Screening for clones with desired DNA inserts and biological properties. These steps are described in some detail in a related article (
molecular cloningMolecular cloning refers to a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms...
).
Expression of recombinant DNA
Following transplantation into the host organism, the foreign DNA contained within the recombinant DNA construct may or may not be
expressedGene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
. That is, the DNA may simply be replicated without expression, or it may be
transcribedTranscription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
and translated so that a recombinant protein is produced. Generally speaking, expression of a foreign gene requires restructuring the gene to include sequences that are required for producing a mRNA molecule that can be used by the host's translational apparatus (e.g. promoter,
translational initiation signalThe Shine-Dalgarno sequence , proposed by Australian scientists John Shine and Lynn Dalgarno , is a ribosomal binding site in the mRNA, generally located 8 basepairs upstream of the start codon AUG. The Shine-Dalgarno sequence exists only in prokaryotes. The six-base consensus sequence is AGGAGG;...
, and
transcriptional terminatorIn genetics, a terminator, or transcription terminator is a section of genetic sequence that marks the end of gene or operon on genomic DNA for transcription.In prokaryotes, two classes of transcription terminators are known:...
). 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
phenotypeA phenotype is an organism's observable characteristics or traits: such as its morphology, development, biochemical or physiological properties, behavior, and products of behavior...
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 or
western hybridizationThe western blot is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide...
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 inactivationIn genetics, an insertion is the addition of one or more nucleotide base pairs into a DNA sequence. This can often happen in microsatellite regions due to the DNA polymerase slipping...
, in which the rDNA becomes inserted into a host cell’s gene. In some cases, researchers use this phenomenon to “
knock outA gene knockout is a genetic technique in which one of an organism's genes is made inoperative . Also known as knockout organisms or simply knockouts, they are used in learning about a gene that has been sequenced, but which has an unknown or incompletely known function...
” genes in order 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 technology
Recombinant DNA is widely used in
biotechnologyBiotechnology is a field of applied biology that involves the use of living organisms and bioprocesses in engineering, technology, medicine and other fields requiring bioproducts. Biotechnology also utilizes these products for manufacturing purpose...
,
medicineMedicine is the science and art of healing. It encompasses a variety of health care practices evolved to maintain and restore health by the prevention and treatment of illness....
and
researchResearch can be defined as the scientific search for knowledge, or as any systematic investigation, to establish novel facts, solve new or existing problems, prove new ideas, or develop new theories, usually using a scientific method...
. Today, recombinant proteins and other products that result from the use of rDNA technology are found in essentially every western pharmacy, doctor's or veterinarian's office, medical testing laboratory, and biological research laboratory. In addition, organisms that have been manipulated using recombinant DNA technology, and products derived from those organisms have found their way into many farms, supermarkets,
home medicine cabinetsHumulin is the brand name for a group of biosynthetic human insulin products, originally developed by Genentech in 1978 and later acquired by Eli Lilly and Company, the company who arguably facilitated...
and even
pet shopsThe GloFish is a patented and trademarked brand of genetically modified fluorescent zebrafish with bright red, green, orange-yellow, blue, and purple fluorescent colors...
.
The most common application of recombinant DNA is in basic research, where it 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 human and veterinary medicine, in agriculture, and in bioengineering. Some specific examples are identified below.
- Recombinant human insulin
Insulin is a hormone central to regulating carbohydrate and fat metabolism in the body. Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle....
. Recombinant insulin has almost completely replaced insulin obtained from animal sources (e.g. pigs and cattle) for the treatment of insulin-dependent diabetes. A variety of different recombinant insulin preparations are in widespread use. DrugBank entry.
- Recombinant human growth hormone
Growth hormone is a peptide hormone that stimulates growth, cell reproduction and regeneration in humans and other animals. Growth hormone is a 191-amino acid, single-chain polypeptide that is synthesized, stored, and secreted by the somatotroph cells within the lateral wings of the anterior...
(HGH, somatotropin). 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 developing Creutzfeldt-Jacob 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 is an essential blood clotting factor also known as anti-hemophilic factor . In humans, Factor VIII is encoded by the F8 gene...
. Recombinant factor VIII is 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 diseasesA blood-borne disease is one that can be spread through contamination by blood.The most common examples are HIV, hepatitis B, hepatitis C and viral hemorrhagic fevers....
, for example HIV and hepatitis B. DrugBank entry
- Recombinant hepatitis B vaccine
Hepatitis B vaccine is a vaccine developed for the prevention of hepatitis B virus infection. The vaccine contains one of the viral envelope proteins, hepatitis B surface antigen . It is produced by yeast cells, into which the genetic code for HBsAg has been inserted...
. Prevention of hepatitis B 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 vitroIn vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
. Vaccine information from Hepatitis B Foundation
- Diagnosis of infection with HIV
Human immunodeficiency virus is a lentivirus that causes acquired immunodeficiency syndrome , a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive...
. Each of the three widely-used methods for diagnosing HIV infectionHIV tests are used to detect the presence of the human immunodeficiency virus , the virus that causes acquired immunodeficiency syndrome , in serum, saliva, or urine. Such tests may detect antibodies, antigens, or RNA.- Terminology :...
has been developed using recombinant DNA. The antibody test (ELISAEnzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...
or western blotThe western blot is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide...
) 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 transcriptase 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 variety of Oryza sativa rice produced through genetic engineering to biosynthesize beta-carotene, a precursor of pro-vitamin A in the edible parts of rice...
is 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 deficiencyVitamin A deficiency is a lack of vitamin A in humans. It is common in developing countries but rarely seen in developed countries. Night blindness is one of the first signs of vitamin A deficiency. Xerophthalmia and complete blindness can also occur since Vitamin A has a major role in...
in the world's population. Golden rice is not currently in use, pending the resolution of intellectual property, environmental and nutritional issues.
- Herbicide-resistant crops Commercial varieties of important agricultural crops (including soy, maize/corn, sorghum, canola, alfalfa and cotton) have been developed which 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 thuringiensis is a Gram-positive, soil-dwelling bacterium, commonly used as a biological pesticide; alternatively, the Cry toxin may be extracted and used as a pesticide. B...
. 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 which 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.
History of recombinant DNA
The idea for 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.
Stanford UniversityThe Leland Stanford Junior University, commonly referred to as Stanford University or Stanford, is a private research university on an campus located near Palo Alto, California. It is situated in the northwestern Santa Clara Valley on the San Francisco Peninsula, approximately northwest of San...
applied for a US patent on recombinant DNA in 1974, listing the inventors as Stanley N. Cohen and Herbert W. Boyer; this patent was awarded in 1980. The first licensed drug generated using recombinant DNA technology was human insulin, developed by
GenentechGenentech Inc., or Genetic Engineering Technology, Inc., is a biotechnology corporation, founded in 1976 by venture capitalist Robert A. Swanson and biochemist Dr. Herbert Boyer. Trailing the founding of Cetus by five years, it was an important step in the evolution of the biotechnology industry...
and Licensed by
Eli Lilly and CompanyEli Lilly and Company is a global pharmaceutical company. Eli Lilly's global headquarters is located in Indianapolis, Indiana, in the United States...
.
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 DNAThe Asilomar Conference on Recombinant DNA was an influential conference organized by Paul Berg discussing the potential biohazards and regulation of biotechnology held in February 1975 at a conference center Asilomar State Beach...
, these concerns were discussed and a voluntary moratorium on recombinant DNA research was initiated for experiments that were thought to be 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 organismsA genetically modified organism or genetically engineered organism is an organism whose genetic material has been altered using genetic engineering techniques. These techniques, generally known as recombinant DNA technology, use DNA molecules from different sources, which are combined into one...
and
genetically-modified food controversiesThe genetically modified foods controversy is a dispute over the relative advantages and disadvantages of genetically modified food crops and other uses of genetically-modified organisms in food production. The dispute involves biotechnology companies, governmental regulators, non-governmental...
.
See also
- Asilomar conference on recombinant DNA
The Asilomar Conference on Recombinant DNA was an influential conference organized by Paul Berg discussing the potential biohazards and regulation of biotechnology held in February 1975 at a conference center Asilomar State Beach...
- Genetic engineering
Genetic engineering, also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest...
- Genetically modified organism
A genetically modified organism or genetically engineered organism is an organism whose genetic material has been altered using genetic engineering techniques. These techniques, generally known as recombinant DNA technology, use DNA molecules from different sources, which are combined into one...
- Recombinant virus
A recombinant virus is a virus produced by recombining pieces of DNA using recombinant DNA technology. This may be used to produce viral vaccines or gene therapy vectors....
- Vector DNA
Further reading
- Judson, Horace F. 1979. The Eighth Day of Creation: Makers of the Revolution in Biology. Touchstone Books, ISBN 0-671-22540-5. 2nd edition: Cold Spring Harbor Laboratory Press, 1996 paperback: ISBN 0-879-69478-5.
- Micklas, David. 2003. DNA Science: A First Course. Cold Spring Harbor Press: ISBN 978-0-879-69636-8.
- Rosenfeld, Israel. 2010. DNA: A Graphic Guide to the Molecule that Shook the World. Columbia University Press: ISBN 978-0-231-14271-7.
- Schultz, Mark and Zander Cannon. 2009. The Stuff of Life: A Graphic Guide to Genetics and DNA. Hill and Wang: ISBN 0-809-08947-5.
- Watson, James. 2004. DNA: The Secret of Life. Random House: ISBN 978-0-099-45184-6.
External links