1. Molecular cloning

Molecular cloning

Main article: Molecular cloning

Molecular cloning refers to the process of making multiple copies of a defined DNA sequence. Cloning is frequently 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 widearray of biological experiments and practical applications ranging fromgenetic 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 regiondoes not necessarily enable one to isolate or amplify the relevantgenomic sequence.

In practice, 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 ofitself and any linked sequence. However, a number of other features areneeded 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 ^[1]

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 anumber of alternative routes can be selected, these are summarized as acloning strategy’.

Initially, the DNA of interest needs to be isolated to provide a DNAsegment of suitable size. Subsequently, a ligation procedure is usedwhere 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 istransfected into cells. A number of alternative techniques areavailable, such as chemical sensitivation of cells, electroporation and biolistics.Finally, the transfected cells are cultured. As the aforementionedprocedures are of particularly low efficiency, there is a need toidentify the cells that have been successfully transfected with thevector construct containing the desired insertion sequence in therequired orientation. Modern cloning vectors include selectable antibioticresistance markers, which allow only cells in which the vector has beentransfected, to grow. Additionally, the cloning vectors may containcolour selection markers which provide blue/white screening (α-factorcomplementation) on X-galmedium. Nevertheless, these selection steps do not absolutely guaranteethat the DNA insert is present in the cells obtained. Furtherinvestigation of the resulting colonies is required to confirm thatcloning was successful. This may be accomplished by means of PCR, restriction fragment analysis and/or DNA sequencing.