Molecular cloning refers to the procedure of isolating a defined DNA sequence and obtaining multiple copies of it in vivo. Cloning is frequently employed to amplify DNA fragments containing genes, but it can be used to amplify any DNA sequence such as promoters, non-coding sequences, chemically synthesised oligonucleotides and randomly fragmented DNA. Cloning is utilized in a wide array of biological experiments and technological applications such as large scale protein production.

Overview

In essence, in order to amplify any DNA sequence in vivo, the sequence in question must be linked to primary sequence elements capable of directing the replication and propagation of themselves and the linked sequence in the desired target host. The required sequence elements differ according to host, but invariably include an origin of replication, and a selectable marker. In practice, however, a number of other features are desired and a variety of specialized cloning vectors exist that allow protein expression, tagging, single stranded RNA and DNA production and a host of other manipulations that are useful in downstream applications.

Recombinase based Cloning

A novel procedure of cloning or subcloning of any DNA fragment by inserting the special DNA fragment of interest into a special area of target DNA through interchange of the relevant DNA fragments.^[1]

This is a one-step reaction: simple, efficient, facilitating high throughput or automatic cloning and/or subcloning.^[2]

Restriction/Ligation Cloning

In the classical restriction and ligation cloning protocols, cloning of any DNA fragment essentially involves four steps: DNA fragmentation with restriction endonucleases, ligation of DNA fragments to a vector, transfection, and screening/selection. Although these steps are invariable among cloning procedures a number of alternative routes can be selected at various points depending on the particular application; these are summarized as a ?cloning strategy?.

Isolation of insert

Initially, the DNA fragment to be cloned needs to be isolated. Preparation of DNA fragments for cloning can be accomplished in a number of alternative ways. Insert preparation is frequently achieved by means of polymerase chain reaction, but it may also be accomplished by restriction enzyme digestion, DNA sonication and fractionation by agarose gel electrophoresis. Chemically synthesized oligonucleotides can also be used if the target sequence size does not exceed the limit of chemical synthesis. Isolation of insert can be done by using shotgun cloning, c-DNA clones, gene machines (artificial chemical synthesis).

Transformation

Following ligation, the ligation product (plasmid) is transformed into bacteria for propagation. The bacteria is then plated on selective agar to select for bacteria that has your plasmid of interest. Individual colonies are picked and tested for the wanted insert. Maxiprep can be done to obtain large quantity of the plasmid containing your inserted gene.

Transfection

Following ligation, a portion of the ligation reaction, including vector with insert in the desired orientation is transfected into cells. A number of alternative techniques are available, such as chemical sensitization of cells, electroporation and biolistics. Chemical sensitization of cells is frequently employed since this does not require specialized equipment and provides relatively high transformation efficiencies. Electroporation is used when extremely high transformation efficiencies are required, as in very inefficient cloning strategies. Biolistics are mainly utilized in plant cell transformations, where the cell wall is a major obstacle in DNA uptake by cells.

Selection

Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low efficiency, there is a need to identify the cells that contain the desired insert at the appropriate orientation and isolate these from those not successfully transformed. Modern cloning vectors include selectable markers (most frequently antibiotic resistance markers) that allow only cells in which the vector, but not necessarily the insert, has been transfected to grow. Additionally, the cloning vectors may contain colour selection markers which provide blue/white screening (via ?-factor complementation) on X-gal medium. Nevertheless, these selection steps do not absolutely guarantee that the DNA insert is present in the cells. Further investigation of the resulting colonies is required to confirm that cloning was successful. This may be accomplished by means of PCR, restriction fragment analysis and/or DNA sequencing.

Blank Cell Cloning

This form of cloning is fairly simple. A nucleus is extracted from a cell, thus leaving the cell with no genetic instructions. After this step, the nucleus of another cell is injected into the cell where the nucleus was removed. The cell receives an electric shock, thus forcing it back to life. The cell can then be forced into rapidly reproducing, thus producing new genetic material.

History Of Cloning

In 1962, biologist John Gurdon of Oxford University announced that he had used the nucleus of fully differentiated adult intestinal cells to clone South African frogs. Gurdon's results electrified the scientific community, but some scientists remained skeptical and began to find flaws in his work.

In 1963, the British biologist J.B.S. Haldane is credited to have coined the term "clone" in a speech entitled "Biological Possibilities for the Human Species of the Next Ten-Thousand Years." Even though many scientists had described, and even completed the cloning process by this time, the term "cloning" had never been used to describe such experiments.

In 1966, Marshall Niremberg, Heinrich Mathaei, and Severo Ochoa crack the genetic code. The cracking of the genetic code opened the door for the explosion of genetic engineering studies and achievements beginning in the late 1970's.

In 1967, the enzyme DNA ligase was isolated. DNA ligase binds together strands of DNA. Its discovery, with the isolation of the first restriction enzyme 1970, paved the way for the first recombinant DNA molecules to be created by Paul Berg in 1972. In the recombinant DNA process, ligase bonds the "sticky" ends of complimentary DNA strands previously cut by a restriction enzyme.

In 1969, James Shapiero of Harvard University, working with Johnathan Beckwith announce that they had isolated the first gene. The gene directed the digestion of sugar in a certain type of bacteria. Shapiero and Beckwith's discovery part of a wave of molecular biology discoveries directly following the 1966 cracking of the genetic code. The announcement also increased the public's concern about the growing power of molecular biologists.

In 1970, both Howard Temin and David Baltimore, working independently, isolated the first restriction enzyme. The restriction enzyme cut DNA molecules at precise locations. This capability led to the future manipulation of DNA.

In 1972, Paul Berg of Stanford University created the first recombinant DNA molecules by combining the DNA of two different organisms.

In 1973, Stanley Cohen and Herbert Boyer created the first recombinant DNA organism using recombinant DNA techniques pioneered a year earlier by Paul Berg. Recombinant DNA, also called gene splicing, is a technique that allows scientists to manipulate the DNA of an organism.

In 1977, German developmental biologist Karl Illmensee, working with Peter Hoppe at Jackson Laboratory in Maine, created mice with only a single parent. They grew mice with only a father as well as mice with only a mother.

In 1979, One of the most surprising of modern genetics announcements was made, Karl Illmensee claimed to have cloned three mice in 1979. The announcement came at a time where a succession of failed cloning attempts were beginning to convince biologists that the cloning of a mammal was impossible.

In 1983, In what has been called by some the greatest achievement of modern molecular biology, Kary B. Mullis developed the polymerase chain reaction (PCR) in 1983. PCR allows the rapid synthesis of designated fragments of DNA. Using the technique, over one billion copies can be synthesized in a matter of hours.

In 1983 Davor Solter, working with David McGrath, attempted to clone mice using his own version of the nuclear transfer method. They wanted to use the cloning experiment to determine if DNA specializes as a cell specializes.

In 1984, Danish scientist Steen Willadsen succeeded in cloning a sheep from embryo cells. His work was the first verified cloning of a mammal using the method of nuclear transfer.

In 1985 Steen Willadsen, the first to clone a farm animal using the nuclear transfer method, joined Grenada Genetics, a bioengineering company. Willadsen used his cloning technique to duplicate the embryos of prize cattle. Grenada Genetics saw the profitability of the future cattle cloning industry. Top breed cattle embryos were highly desired by farmers, Willadsen's procedure mass produced identical copies of such embryos.

In 1986, while working at Grenada Genetics, Steen Willadsen cloned a cow using differentiated, one week old embryo cells. The work proved that the genetic information of a cell did not diminish as a cell specialized and that DNA could return to its original state. Willadsen never officially published his results, but the work was a strong influence in Ian Wilmut's decision to attempt to clone from adult cells, which he accomplished in the famous 1996 birth of "Dolly."

In 1986, Neal First, Randal Prather, and Willard Eyestone, working at the University of Wisconsin, cloned a cow from early embryo cells. Though the race to clone the first farm animal had already been won by Steen Willadsen in 1984, Prather, Eyestone, and First's project was undertaken roughly at the same time as Willadsen's.

In October of 1990, the National Institutes of Health officially began the Human Genome Project, a massive international collaborative effort to locate the 50,000 to 100,000 genes and sequence the estimated 3 billion nucleotides making up the entire human genome. By determining the complete genetic sequence, scientists hope to begin correlating human traits with certain genes. With this information, medical researchers have begun to determine the intricacies of human gene function, including the source of genetic disorders and diseases that have plagued medical researchers for years.

In July 1995, Ian Wilmut and Keith Campbell of the Roslin Institute in Scotland successfully cloned two sheep, named Megan and Morag, from differentiated embryo cells. The idea to clone sheep was arrived at by Ian Wilmut as an answer to a gene insertion project he was researching. At the time, time inserting genes into embryo cells was a difficult and tedious process. Few embryos survived the insertion of a gene, even fewer incorporated the gene into their genetic code, and even fewer organisms developed properly and used the gene in all of their cells.

On July 5, 1996, Dolly, the first organism ever to be cloned from adult cells, was born. Ian Wilmut and Keith Campbell, researchers at the Roslin Institute in Scotland created Dolly using a technique similar to that with which they created the first sheep from differentiated embryo cells in 1995.

On March 4, 1997, President Clinton, in response to the large scale human cloning ethics debate brought about by Ian Wilmut's announcement of the creation of Dolly, proposed a five year moratorium on federal and privately funded human cloning research. In addition to this proposal, Clinton asked the National Bioethics Advisory Commission to review the prospects of human cloning and determine if legal preventive actions should be taken.

On December 5, 1997, Harvard graduate Richard Seed announced that he planned to clone a human being before any federal laws could be enacted to ban the process. Seed's announcement added fuel to the raging ethical debate on human cloning that had been sparked by Ian Wilmut's creation of Dolly, the first clone obtained from adult cells.

In July 1997, building upon their success of the creation of Dolly, the first animal cloned from adult cells, Ian Wilmut and Keith Campbell created Polly, a Poll Dorset lamb cloned from skin cells grown in a lab and genetically altered to contain a human gene. Polly's birth signified the first step in the application of cloning technology to the production of a useful product. Most scientists believe that most beneficial application of cloning will come from the exact reproduction of animals genetically altered to produce human proteins or organs more easily accepted in transplants. Wilmut and Campbell's creation of Polly surprised the scientific community by how fast cloning technology was progressing. The cloning of genetically altered farm animals was not expected for another five years.

In July 1998, Ryuzo Yanagimachi, Toni Perry, and Teruhiko Wakayama of the University of Hawaii announced that they had cloned fifty mice from adult cells since October of 1997. The new cloning technique, which has proven to be more efficient than that performed by Ian Wilmut in his cloning of Dolly, was developed by postdoctoral student Wakayama in his spare time.

Information taken from the site: http://home.hawaii.rr.com/johns/history.htm

References

• Copeland NG, Jenkins NA, Court DL. Recombineering: a powerful new tool for mouse functional genomics. Nat Rev Genet. 2001 Oct;2(10):769-79. Review.
• Lu JP, Beatty LK, Pinthus JH. Dual expression recombinase based (DERB) single vector system for high throughput screening and verification of protein interactions in living cells.". Nature Precedings 2008 http://hdl.handle.net/10101/npre.2008.1550.2

See also

• Cloning
• Subcloning
• Polymerase chain reaction

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