Cloning is the process of creating an identical copy of something. In , it collectively refers to processes used to create copies of fragments (Molecular Cloning), (Cell Cloning), or . The term also encompases situations, whereby organisms reproduce , but in common parlance refers to intentionally created copies of organisms.
The term clone is derived from клщн, the word for "twig", referring to the process, whereby a new plant can be created from a twig. In , the spelling clon was used until the twentieth century; the final e came into use to indicate the vowel is a "long o" instead of a "short o". Since the term entered the popular lexicon in a more general context, the spelling clone has been used exclusively.
Molecular cloning refers to the procedure of isolating a defined DNA sequence and obtaining multiple copies of it . Cloning is frequently employed to amplify DNA fragments containing , but it can be used to amplify any DNA sequence such as , non-coding sequences and randomly fragmented DNA. It is utilised in a wide array of biological experiments and practical applications such as large scale protein production. Occasionally, the term cloning is misleadingly used to refer to the identification of the 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 essence, in order to amplify any DNA sequence in a living organism that sequence must be linked to an , a sequence element capable of directing the propagation of its self and any linked sequence. In practice, however, a number of other features are desired and a variety of specialised cloning exist that allow protein expression, tagging, single stranded and DNA production and a host of other manipulations.
Cloning of any DNA fragment essentially involves four steps: fragmentation, ligation, transfection, and screening/selection. Although these steps are invariable among cloning procedures a number of alternative routes can be selected, these are summarised as a ‘cloning strategy’.
Initially, the DNA of interest needs to be isolated to provide a relevant DNA segment of suitable size. Subsequently, a ligation procedure is employed whereby the amplified fragment is inserted into a vector. The vector (which is frequently circular) is linearised by means of restriction enzymes, and incubated with the fragment of interest under appropriate conditions with an enzyme called . 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 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 (б-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 .
Cloning a cell means to derive a (clonal) population of cells from a single cell. In the case of unicellular organisms such as bacteria and yeast, this process is remarkably simple and essentially only requires the of the appropriate medium. However, in the case of cell cultures from higher organisms, cell cloning is an arduous task as these cells will not readily grow in standard media.