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Cloning, Cloning

Cloning in biotechnology refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms. More generally, the term refers to the production of multiple copies of a product such as digital media or software.

It is used in a wide array of biological experiments and practical applications ranging from genetic fingerprinting to large scale protein production. Occasionally, the term cloning is misleadingly used to refer to the identification of the chromosomal location of a gene associated with a particular phenotype of interest, such as in positional cloning. In practice, localization of the gene to a chromosome or genomic region does not necessarily enable one to isolate or amplify the relevant genomic sequence. In order to amplify any DNA sequence in a living organism, that sequence must be linked to an origin of replication, which is a sequence of DNA capable of directing the propagation of itself and any linked sequence. However, a number of other features are needed and a variety of specialised cloning vectors (small piece of DNA into which a foreign DNA fragment can be inserted) exist that allow protein expression, tagging, single stranded RNA and DNA production and a host of other manipulations.

The vector (which is frequently circular) is linearised using restriction enzymes, and incubated with the fragment of interest under appropriate conditions with an enzyme called DNA ligase. Following ligation the vector with the insert of interest is transfected into cells. A number of alternative techniques are available, such as chemical sensitivation of cells, electroporation and biolistics. Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low efficiency, there is a need to identify the cells that have been successfully transfected with the vector construct containing the desired insertion sequence in the required orientation. Modern cloning vectors include selectable antibiotic resistance markers, which allow only cells in which the vector has been transfected, to grow. Additionally, the cloning vectors may contain colour selection markers which provide blue/white screening (-factor complementation) on X-gal medium. Nevertheless, these selection steps do not absolutely guarantee that the DNA insert is present in the cells obtained. 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.

However, in the case of cell cultures from multi-cellular organisms, cell cloning is an arduous task as these cells will not readily grow in standard media.

At an early growth stage when colonies consist of only a few of cells, sterile polystyrene rings (cloning rings), which have been dipped in grease are placed over an individual colony and a small amount of trypsin is added. Cloned cells are collected from inside the ring and transferred to a new vessel for further growth.

This process is also called "research cloning" or "therapeutic cloning." The goal is not to create cloned human beings, but rather to harvest stem cells that can be used to study human development and to potentially treat disease. While a clonal human blastocyst has been created, stem cell lines are yet to be isolated from a clonal source.

Many horticultural plant cultivars are clones, having been derived from a single individual, multiplied by some process other than sexual reproduction. As an example, some European cultivars of grapes represent clones that have been propagated for over two millennia. Other examples are potato and banana. Grafting can be regarded as cloning, since all the shoots and branches coming from the graft are genetically a clone of a single individual, but this particular kind of cloning has not come under ethical scrutiny and is generally treated as an entirely different kind of operation.

Scientists speculated that Dolly's death in 2003 was related to the shortening of telomeres, DNA-protein complexes that protect the end of linear chromosomes. However, other researchers, including Ian Wilmut who led the team that successfully cloned Dolly, argue that Dolly's early death due to respiratory infection was unrelated to deficiencies with the cloning process.

The term is generally used to refer to artificial human cloning; human clones in the form of identical twins are commonplace, with their cloning occurring during the natural process of reproduction. There are two commonly discussed types of human cloning: therapeutic cloning and reproductive cloning. Therapeutic cloning involves cloning cells from an adult for use in medicine and is an active area of research: while reproductive cloning would involve making cloned human beings. Such reproductive cloning has not been performed and is illegal in many countries. A third type of cloning called replacement cloning is a theoretical possibility, and would be a combination of therapeutic and reproductive cloning. Replacement cloning would entail the replacement of an extensively damaged, failed, or failing body through cloning followed by whole or partial brain transplant.

Dr. Samuel Wood and a colleague donated skin cells, and DNA from those cells was transferred to human eggs. It is not clear if the embryos produced would have been capable of further development, but Dr. Wood stated that if that were possible, using the technology for reproductive cloning would be both unethical and illegal. The 5 cloned embryos, created in Stemagen Corporation lab, in La Jolla, were destroyed.

Others have countered that people who exist today and have interpersonal relationships and personal histories should take precedence over never-conscious life at any stage of developmental maturity. The Catholic Church and many religious organizations oppose all forms of cloning, on the grounds that life begins at conception. Conversely, Judaism does not equate life with conception and, though some question the wisdom of cloning, Orthodox rabbis generally find no firm reason in Jewish law and ethics to object to cloning. Michael Brody. Avraham Steinberg.

In real life, one of the most anticipated targets for cloning was once the Woolly Mammoth, but attempts to extract DNA from frozen mammoths have been unsuccessful, though a joint Russo-Japanese team is currently working toward this goal.

These successes provided hope that similar techniques (using surrogate mothers of another species) might be used to clone extinct species. Anticipating this possibility, tissue samples from the last bucardo (Pyrenean Ibex) were frozen immediately after it died. Researchers are also considering cloning endangered species such as the giant panda, ocelot, and cheetah. The "Frozen Zoo" at the San Diego Zoo now stores frozen tissue from the world's rarest and most endangered species.

Cloning from a single specimen could not create a viable breeding population in sexually reproducing animals. Furthermore, even if males and females were to be cloned, the question would remain open whether they would be viable at all in the absence of parents that could teach or show them their natural behavior. Essentially, if cloning an extinct species were successful it must be considered that cloning is still an experimental technology that succeeds only by chance it is more likely than not that any resulting animals, even if they were healthy, would be little more than curios or museum pieces.

Many conservation biologists and environmentalists vehemently oppose cloning endangered species mainly because they think it may deter donations to help preserve natural habitat and wild animal populations. The "rule-of-thumb" in animal conservation is that, if it is still feasible to conserve habitat and viable wild populations, breeding in captivity should not be undertaken in isolation.

Source: Wikipedia > Cloning