Recombinant DNA

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]

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.

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Introduction

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

Cloning and relation to plasmids

Main article: Cloning

  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] As a general definition of plasmids, the definition is that they 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

Main article: Chimeric DNA

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 complimentary 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 rDNA

In the 1920s, there was no known way to produce insulin. The problem was solved, temporarily at least, when it was found that insulin from the pancreas of a pig could be used in humans. This method was the primary solution for diabetes until about six years ago. The problem with insulin production was raised again: There were not enough pigs to keep the supply of insulin intact. Scientists needed to devise another way. This is where one of the biggest breakthroughs in recombinant DNA technology happened.

The specific gene sequence, or oligonucleotide, that codes for insulin production in humans was introduced to a sample colony of E. coli. Only about 1 out of 10⁶ bacteria picks up the sequence. This is not really a problem, however, because the growth rate is only about 30 minutes for E. coli. In a 24-hour period, there may be billions of E. coli that are coded with the DNA sequences needed to induce insulin production.^[4]

See also

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

Notes

References