Recombinant DNA

Recombinant DNA (rDNA) molecules are DNA sequences that result from the use of laboratory methods (molecular cloning) to bring together genetic material from multiple sources, creating sequences 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 replication 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 chimera.

The DNA sequences used in the construction of recombinant DNA molecules can originate from any species. 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 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^{[citation needed]}. Expression of foreign proteins requires the use of specialized expression vectors and often necessitates significant restructuring of the foreign coding sequence^{[citation needed]}.

It is important to note that 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.

Creating recombinant DNA

Main article: Molecular cloning

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.

Molecular cloning is the laboratory process used to create recombinant DNA.^[1][2][3][4] It is one of two widely-used methods (along with polymerase chain reaction, 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 vector, a DNA molecule that will replicate 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.^[5] 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, (7) Screening for clones with desired DNA inserts and biological properties.^[4] These steps are described in some detail in a related article (molecular cloning).

Expression of recombinant DNA

Main article: Gene 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 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 signal, and transcriptional terminator).^[6] 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.^[7]

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.^[8] 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.^[8] 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.^[9] 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 in order to determine their biological function and importance.^[10] 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 biotechnology, medicine and research. 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 cabinets and even pet shops.

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.^[8] 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.^[2]

Many additional practical applications of recombinant DNA are found in human and veterinary medicine, in agriculture, and in bioengineering.^[2] Some specific examples are identified below.

• Recombinant human insulin. 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.^[11] DrugBank entry.
• Recombinant human growth hormone (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.^[12] It has also been misused as a performance enhancing drug by athletes and others.^[13] DrugBank entry
• Recombinant blood clotting factor VIII. 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.^[14] 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. 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 vitro. Vaccine information from Hepatitis B Foundation
• 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 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 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 is a recombinant variety of rice that has been engineered to express the enzymes responsible for β-carotene biosynthesis.^[9] This variety of rice holds substantial promise for reducing the incidence of vitamin A deficiency in the world's population.^[15] 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.^[16] 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.^[15] 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.^[17]

History of recombinant DNA

Main article: History of biotechnology

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.^[18] The first publications describing the successful production and intracellular replication of recombinant DNA appeared in 1972 and 1973.^[19][20][21] Stanford University 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.^[22] The first licensed drug generated using recombinant DNA technology was human insulin, developed by Genentech and Licensed by Eli Lilly and Company.^[23]

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 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 organisms and genetically-modified food controversies.

See also

• Asilomar conference on recombinant DNA
• Genetic engineering
• Genetically modified organism
• Recombinant virus
• Vector DNA

References

1. ^ Campbell, Neil A. & Reece, Jane B.. (2002). Biology (6th ed.). San Francisco: Addison Wesley. pp. 375–401. ISBN 0-201-75054-6. 
2. ^ ^{a b c} Peter Walter; Alberts, Bruce; Johnson, Alexander S.; Lewis, Julian; Raff, Martin C.; Roberts, Keith (2008). Molecular Biology of the Cell (5th edition, Extended version). New York: Garland Science. ISBN 0-8153-4111-3. . Fourth edition is available online through the NCBI Bookshelf: link
3. ^ Berg, Jeremy Mark; Tymoczko, John L.; Stryer, Lubert (2010). Biochemistry, 7th ed. (Biochemistry (Berg)). W.H. Freeman & Company. ISBN 1-4292-2936-5.  Fifth edition available online through the NCBI Bookshelf: link
4. ^ ^{a b} Watson, James D. (2007). Recombinant DNA: Genes and Genomes: A Short Course. San Francisco: W.H. Freeman. ISBN 0-7167-2866-4. 
5. ^ Russell, David W.; Sambrook, Joseph (2001). Molecular cloning: a laboratory manual. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory. ISBN 0-87969-576-5. 
6. ^ Hannig, G.; Makrides, S. (1998). "Strategies for optimizing heterologous protein expression in Escherichia coli". Trends in biotechnology 16 (2): 54–60. PMID 9487731.  edit
7. ^ Brondyk, W. H. (2009). "Chapter 11 Selecting an Appropriate Method for Expressing a Recombinant Protein". Methods in enzymology 463: 131–147. doi:10.1016/S0076-6879(09)63011-1. PMID 19892171.  edit
8. ^ ^{a b c} Brown, Terry (2006). Gene Cloning and DNA Analysis: an Introduction. Cambridge, MA: Blackwell Pub. ISBN 1-4051-1121-6. 
9. ^ ^{a b} Ye, X.; Al-Babili, S.; Klöti, A.; Zhang, J.; Lucca, P.; Beyer, P.; Potrykus, I. (2000). "Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm". Science 287 (5451): 303–305. doi:10.1126/science.287.5451.303. PMID 10634784.  edit
10. ^ Koller, B. H.; Smithies, O. (1992). "Altering Genes in Animals by Gene Targeting". Annual Review of Immunology 10: 705–730. doi:10.1146/annurev.iy.10.040192.003421. PMID 1591000.  edit
11. ^ Gualandi-Signorini, A.; Giorgi, G. (2001). "Insulin formulations--a review". European review for medical and pharmacological sciences 5 (3): 73–83. PMID 12004916.  edit
12. ^ Von Fange, T.; McDiarmid, T.; MacKler, L.; Zolotor, A. (2008). "Clinical inquiries: Can recombinant growth hormone effectively treat idiopathic short stature?". The Journal of family practice 57 (9): 611–612. PMID 18786336.  edit
13. ^ Fernandez, M.; Hosey, R. (2009). "Performance-enhancing drugs snare nonathletes, too". The Journal of family practice 58 (1): 16–23. PMID 19141266.  edit
14. ^ Manco-Johnson, M. J. (2010). "Advances in the Care and Treatment of Children with Hemophilia". Advances in Pediatrics 57 (1): 287–294. doi:10.1016/j.yapd.2010.08.007. PMID 21056743.  edit
15. ^ ^{a b} Paine, J. A.; Shipton, C. A.; Chaggar, S.; Howells, R. M.; Kennedy, M. J.; Vernon, G.; Wright, S. Y.; Hinchliffe, E. et al. (2005). "Improving the nutritional value of Golden Rice through increased pro-vitamin a content". Nature Biotechnology 23 (4): 482–487. doi:10.1038/nbt1082. PMID 15793573.  edit
16. ^ Funke, T.; Han, H.; Healy-Fried, M.; Fischer, M.; Schönbrunn, E. (2006). "Molecular basis for the herbicide resistance of Roundup Ready crops". Proceedings of the National Academy of Sciences 103 (35): 13010–13015. doi:10.1073/pnas.0603638103. PMC 1559744. PMID 16916934. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=1559744.  edit
17. ^ Mendelsohn, M.; Kough, J.; Vaituzis, Z.; Matthews, K. (2003). "Are Bt crops safe?". Nature Biotechnology 21 (9): 1003–1009. doi:10.1038/nbt0903-1003. PMID 12949561.  edit
18. ^ Lear, J. (1978). Recombinant DNA: The Untold Story. New York: Crown Publishers. p. 43.
19. ^ Jackson, D.; Symons, R.; Berg, P. (1972). "Biochemical method for inserting new genetic information into DNA of Simian Virus 40: Circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli". Proceedings of the National Academy of Sciences of the United States of America 69 (10): 2904–2909. doi:10.1073/pnas.69.10.2904. PMC 389671. PMID 4342968. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=389671.  edit
20. ^ Lobban, P.; Kaiser, A. (1973). "Enzymatic end-to end joining of DNA molecules". Journal of molecular biology 78 (3): 453–471. doi:10.1016/0022-2836(73)90468-3. PMID 4754844.  edit
21. ^ Cohen, S.; Chang, A.; Boyer, H.; Helling, R. (1973). "Construction of biologically functional bacterial plasmids in vitro". Proceedings of the National Academy of Sciences of the United States of America 70 (11): 3240–3244. doi:10.1073/pnas.70.11.3240. PMC 427208. PMID 4594039. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=427208.  edit
22. ^ Hughes, S. (2001). "Making dollars out of DNA. The first major patent in biotechnology and the commercialization of molecular biology, 1974-1980". Isis; an international review devoted to the history of science and its cultural influences 92 (3): 541–575. PMID 11810894.  edit
23. ^ Johnson, I. S. (1983). "Human insulin from recombinant DNA technology". Science 219 (4585): 632–637. doi:10.1126/science.6337396. PMID 6337396.  edit

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

• Recombinant DNA fact sheet^{[dead link]} (from University of New Hampshire)
• Plasmids in Yeasts (Fact sheet from San Diego State University)
• Animation illustrating construction of recombinant DNA and foreign protein production by recombinant bacteria
• Recombinant DNA research at UCSF and commercial application at Genentech Edited transcript of 1994 interview with Herbert W. Boyer, Living history project. Oral history.