Unicellular organisms

Cloning

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For the cloning of human beings, see Human cloning. For other uses, see Cloning (disambiguation).

The sea anemone, Anthopleura elegantissima in process of cloning

Cloning in biology is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms. The term also refers to the production of multiple copies of a product such as digital media or software.

The term clone is derived from κλῶνος, the Greek word for "trunk, branch", referring to the process whereby a new plant can be created from a twig. In horticulture, 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".^[1][2] Since the term entered the popular lexicon in a more general context, the spelling clone has been used exclusively.

Molecular cloning

Main article: Molecular cloning

Molecular cloning refers to the process of making multiple molecules. Cloning is commonly used to amplify DNA fragments containing whole genes, but it can also be used to amplify any DNA sequence such as promoters, non-coding sequences and randomly fragmented DNA. 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. 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.

Cloning of any DNA fragment essentially involves four steps^[3]

1. fragmentation - breaking apart a strand of DNA
2. ligation - gluing together pieces of DNA in a desired sequence
3. transfection - inserting the newly formed pieces of DNA into cells
4. screening/selection - selecting out the cells that were successfully transfected with the new DNA

Although these steps are invariable among cloning procedures a number of alternative routes can be selected, these are summarized as a 'cloning strategy'.

Initially, the DNA of interest needs to be isolated to provide a DNA segment of suitable size. Subsequently, a ligation procedure is used where the amplified fragment is inserted into a vector (piece of DNA). 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, optical injection 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 must be required to confirm that cloning was successful. This may be accomplished by means of PCR, restriction fragment analysis and/or DNA sequencing.

Cellular cloning

Unicellular organisms

Cloning cell-line colonies using cloning rings

Cloning a cell means to derive a 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 inoculation of the appropriate medium. 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.

A useful tissue culture technique used to clone distinct lineages of cell lines involves the use of cloning rings (cylinders).^[4] According to this technique, a single-cell suspension of cells that have been exposed to a mutagenic agent or drug used to drive selection is plated at high dilution to create isolated colonies; each arising from a single and potentially clonal distinct cell. 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.

Date: 2015-12-24; view: 1028