Recombinant DNA

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Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1]

Associate Editor-In-Chief: Cafer Zorkun, M.D., Ph.D. [2]

Recombinant DNA is a form of artificial DNA that is engineered through the combination or insertion of one or more DNA strands, thereby combining DNA sequences that would not normally occur together.[1] In terms of genetic modification, recombinant DNA is produced through the addition of relevant DNA into an existing organismal genome, such as the plasmid of bacteria, to code for or alter different traits for a specific purpose, such as immunity.[1] It differs from genetic recombination, in that it does not occur through processes within the cell or ribosome, but is exclusively engineered.[1]

GloFish are a type of zebrafish with recombinant DNA. Genes for fluorescent proteins have been inserted into their genome to produce their fluorescent colors.

The Recombinant DNA technique was engineered by Stanley Norman Cohen and Herbert Boyer in 1973. They published their findings in a 1974 paper entitled "Construction of Biologically Functional Bacterial Plasmids in vitro", which described a technique to isolate and amplify genes or DNA segments and insert them into another cell with precision, creating a transgenic bacterium. Recombinant DNA technology was made possible by the discovery of restriction endonucleases by Werner Arber, Daniel Nathans, and Hamilton Smith, for which they received the 1978 Nobel Prize in Medicine.

## Introduction

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Because of the importance of DNA in the replication of new structures and characteristics of living organisms, it has widespread importance in recapitulating via viral or non-viral vectors, both desirable and undesirable characteristics of a species to achieve characteristic change or to counteract effects caused by genetic or imposed disorders that have effects upon cellular or organismal processes.[2] Through the use of recombinant DNA, genes that are identified as important can be amplified and isolated for use in other species or applications, where there may be some form of genetic illness or discrepancy, and provides a different approach to complex biological problem solving.[2]

## Applications and methods

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### Cloning and relation to plasmids

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Main article: Cloning

A simple example of how a desired gene is inserted into a plasmid. In this example, the gene specified in the white color becomes useless as the new gene is added.

The use of cloning is interrelated with Recombinant DNA in classical biology, as the term "clone" refers to a cell or organism derived from a parental organism,[1] with modern biology referring to the term as a collection of cells derived from the same cell that remain identical.[1] In the classical instance, the use of recombinant DNA provides the initial cell from which the host organism is then expected to recapitulate when it undergoes further cell division, with bacteria remaining a prime example due to the use of viral vectors in medicine that contain recombinant DNA inserted into a structure known as a plasmid.[1]

Plasmids are extrachromosomal self-replicating circular forms of DNA present in most bacteria, such as Escherichia coli (E. Coli), containing genes related to catabolism and metabolic activity,[1] and allowing the carrier bacterium to survive and reproduce in conditions present within other species and environments. These genes represent characteristics of resistance to bacteriophages and antibiotics[1] and some heavy metals, but can also be fairly easily removed or separated from the plasmid by restriction endonucleases,[1], which regularly produce "sticky ends" and allow the attachment of a selected segment of DNA, which codes for more "reparative" substances, such as peptide hormone medications including insulin, growth hormone, and oxytocin. In the introduction of useful genes into the plasmid, the bacteria are then used as a viral vector, which are encouraged to reproduce so as to recapitulate the altered DNA within other cells it infects, and increase the amount of cells with the recombinant DNA present within them.

The use of plasmids is also key within gene therapy, where their related viruses are used as cloning vectors or carriers, which are means of transporting and passing on genes in recombinant DNA through viral reproduction throughout an organism.[1] Plasmids contain three common features -- a replicator, selectable marker and a cloning site.[1] The replicator or "ori"[1] refers to the origin of replication with regard to location and bacteria where replication begins. The marker refers to a gene that usually contains resistance to an antibiotic, but may also refer to a gene that is attached alongside the desired one, such as that which confers luminescence to allow identification of successfully recombined DNA.[1] The cloning site is a sequence of nucleotides representing one or more positions where cleavage by restriction endonucleases occurs.[1] Most eukaryotes do not maintain canonical plasmids; yeast is a notable exception.[3] In addition, the Ti plasmid of the bacterium Agrobacterium tumefaciens can be used to integrate foreign DNA into the genomes of many plants. Other methods of introducing or creating recombinant DNA in eukaryotes include homologous recombination and transfection with modified viruses.

### Chimeric plasmids

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An example of chimeric plasmid formation from two "blunt ends" via the enzyme, T4 Ligase.

When recombinant DNA is then further altered or changed to host additional strands of DNA, the molecule formed is referred to as "chimeric" DNA molecule,[1] with reference to the mythological chimera, which consisted as a composite of several animals.[1] The presence of chimeric plasmid molecules is somewhat regular in occurrence, as, throughout the lifetime of an organism[1], the propagation by vectors ensures the presence of hundreds of thousands of organismal and bacterial cells that all contain copies of the original chimeric DNA.[1]

In the production of chimeric plasmids, the processes involved can be somewhat uncertain[1], as the intended outcome of the addition of foreign DNA may not always be achieved and may result in the formation of unusable plasmids. Initially, the plasmid structure is linearised[1] to allow the addition by bonding of complementary foreign DNA strands to single-stranded "overhangs"[1] or "sticky ends" present at the ends of the DNA molecule from staggered, or "S-shaped" cleavages produced by restriction endonucleases.[1]

A common vector used for the donation of plasmids originally was the bacterium Escherichia coli and, later, the EcoRI derivative[2], which was used for its versatility[2] with addition of new DNA by "relaxed" replication when inhibited by chloramphenicol and spectinomycin, later being replaced by the pBR322 plasmid.[2]In the case of EcoRI, the plasmid can anneal with the presence of foreign DNA via the route of sticky-end ligation, or with "blunt ends" via blunt-end ligation, in the presence of the phage T4 ligase [2], which forms covalent links between 3-carbon OH and 5-carbon PO4 groups present on blunt ends.[2] Both sticky-end, or overhang ligation and blunt-end ligation can occur between foreign DNA segments, and cleaved ends of the original plasmid depending upon the restriction endonuclease used for cleavage.[2]

### Synthetic insulin production using recombinant DNA

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Until the 1920s, there was no known way to produce insulin because the hormone was not officially identified until 1921. Once identified, the production problem was quickly solved when it was found that insulin from the pancreas of a cow, pig or even some species of fish could be used successfully in humans. This method was the primary solution for type 1 diabetes mellitus for decades, and manufacturing methods had steadily improved the purity of the hormone which was made from animal pancreases. However, proponents of the genetic engineering technology continued to raise what they claimed was a looming problem with traditional methods of insulin production: a supposed shortage of supply in the not-too-distant future. But in the 1987 book "Invisible Frontiers: The Race to Synthesize a Human Gene"[4], author Stephen S. Hall wrote that the supposed shortage is now known to be an assumption based on mistaken facts. He wrote:

> To hear some tell it, there was never a supply problem with pig pancreases in the first place. "The whole thing was rubbish," insists Paul Haycook, research director at Squibb-Novo. "There was never a shortage of pig pancreases, and there never will be." Haycook blames the scare on a miscalculation by an official who had prepared projections for the Food and Drug Administration — a mistake based, ironically, on a mistake in an Eli Lilly training brochure which confused kilograms with pounds. Instead of projecting an insulin shortage by 1982, a revised FDA report predicted adequate insulin supplies through the year 2006. In any event, there is never likely to be a shortage caused by a scarcity of pancreases.[4]

Scientists and entrepreneurs were very eager to prove they could devise another way to synthesize the hormone, in part, because of competition from other researchers and also because of the promise for the fame and fortune that its so-called "discovery" could bring them. Insulin was part of a wider vision to introduce biotechnology medicines, and was chosen specifically because it is a simple hormone and was therefore relatively easy to copy. However, the motive was never to improve the lives of people with diabetes, but to prove that the technology worked. Insulin was chosen as the ideal candidate because it is a relatively simple protein, it was so widely used that if researchers could prove that biosynthetic "human" insulin was safe and effective, then the technology would be accepted as such, and it would open the flood gates for many other products to be made this way, along with millions of dollars.

That was exactly what happened. One of the biggest breakthroughs in recombinant DNA technology happened in the manufacture of biosynthetic "human" insulin, which was the first medicine made via recombinant DNA technology ever to be approved by the FDA.

Henry I. Miller was an early advocate of biotechnology and drugs, and continues to be so as a fellow at the Hoover Institute even though he no longer works for the FDA. Miller began work for the FDA as head of a special department created to establish new procedures for approving drugs created through biotechnology. In his book "To America's Health: A Model for Reform of the Food and Drug Administration" (Hoover Institution Press, 2000), Miller states that he pushed for rapid approval of biosynthetic insulin from his boss, who was not comfortable approving it on such short notice, especially when it had been tested on so few people. Amazingly, Miller admits that he actually waited for his boss to go on vacation, and then took the approval to his boss' boss, who then approved the drug[5].

As far as technical details, the specific gene sequence, or oligonucleotide, that codes for insulin production in humans was introduced to a sample colony of E. coli (the bacteria found in feces). Only about 1 out of 106 bacteria picks up the sequence. However, this is not really a problem, because the lifecycle is only about 30 minutes for E. coli. This means that in a 24-hour period, there may be billions of E. coli that are coded with the DNA sequences needed to induce insulin production.[6]

However, a sampling of initial reaction showed that Humulin was greeted more as a technological rather than a medical breakthrough, and that this sentiment was building even before the drug reached pharmacies. As early as 1980, the British magazine New Scientist reported, "Other big chemical manufacturers predict that Eli Lilly's massive $40 million investment in two plants to make insulin - may be a classic example of backing a loser."

The Economist concluded: "The first bug-built drug for human use may turn out to be a commercial flop. But the way has now been cleared-and remarkably quickly, too - for biotechnologists with interesting new products to clear the regulatory hurdles and run away with the prizes."

Ultimately, widespread consumer adoption of biosynthetic "human" insulin did not occur until the manufacturers removed highly-purified animal insulin from the market.

## See also

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* Asilomar conference on recombinant DNA
* Vector DNA
* List of recombinant proteins
* Transgenic bacteria
* Ice-minus bacteria

## Notes

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1. ↑ 1.00 1.01 1.02 1.03 1.04 1.05 1.06 1.07 1.08 1.09 1.10 1.11 1.12 1.13 1.14 1.15 1.16 1.17 1.18 1.19 1.20 1.21 Jeremy M. Berg, John L. Tymoczko, Lubert Stryer,. Biochemistry. San Francisco: W. H. Freeman. ISBN 0-7167-8724-5.CS1 maint: Multiple names: authors list (link)
2. ↑ 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 Nathan P. Kaplan, Nathan P. Colowick, Ray Wu (1980). Recombinant DNA, Volume 68: Volume 68: Recombinant Dna Part F (Methods in Enzymology). Academic Press. ISBN 0-1218-1968-X.CS1 maint: Multiple names: authors list (link)
3. ↑ "Plasmids in eukaryotic microbes: an example". Retrieved 2007-06-05.
4. ↑ 4.0 4.1 Invisible Frontiers: The Race to Synthesize a Human Gene"(1987, Tempus Books of Microsoft Press)
5. ↑ Miller, Henry I.; "The Drug Bureaucracy"; National Review; Feb 13, 2003.
6. ↑ Human insulin from recombinant DNA technology - Johnson 219 (4585): 632 - Science

## References

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* Garret, R. H.; Grisham, C. M. (2000), Biochemistry, Saunders College Publishers, ISBN 0030758173
* Colowick, S. P.; Kapian, O. N. (1980), Methods in Enzymology - Volume 68; Recombinant DNA, Academic Press, ISBN 012181968X.

## External links

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* Fact Sheet Describing Recombinant DNA and Elements Utilizing Recombinant DNA Such as Plasmids and Viral Vectors, and the Application of Recombinant DNA Techniques in Molecular Biology
* Plasmids in Yeasts
* A 3D animation illustrating the process by which a protein is mass-produced using spliced DNA and bacterial replication

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