Clone (genetics)
From Wikipedia, the free encyclopedia
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 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 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 essence, in order to amplify any DNA sequence in a living organism that sequence must be linked to an origin of replication, a sequence element capable of directing the propagation of itself and any linked sequence. In practice, however, a number of other features are desired and a variety of specialised cloning vectors 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: 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. Preparation of DNA fragments for cloning is frequently achieved by means of PCR, but it may also be accomplished by restriction enzyme digestion, DNA sonication and fractionation by agarose gel electrophoresis.
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 DNA ligase. Ligation procedures usually employ sticky ends, single stranded DNA overhangs which allow annealing of the insert with the vector sequence. Sticky ends can be incorporated into inserts either by chemical modification and attachment of adapter molecules or by incorporation of restriction enzyme recognition sequences into PCR primers and digestion of PCR products with the appropriate restriction enzyme prior to ligation. ‘Sticky ends’ allow for both higher efficiency transformations and directional insertion of the insert into the vector, thus minimising the need for subsequent screening.
Following ligation, the vector with the insert of interest 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 specialised equipment and provides relatively high transformation efficiencies. Electroporation is employed when extremely high transformation efficiencies are required, as in very inefficient cloning strategies. Biolistics are mainly used in plant cell transformations, where the cell wall is a major obstacle in DNA uptake by cells.
Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low efficiency, there is a need to identify the required cells and separate these from those not successfully transformed. The required cells will be those 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 selection marker, which allow only cells in which the vector has been transfected, too grow. Additionally, the cloning vectors may contain colour selection markers which provide blue/white screening (α-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.
