Molecular cloning

[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, chemically synthesised oligonucleotides and randomly fragmented DNA. Cloning is utilized in a wide array of biological experiments and technological applications such as large scale protein production.

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Overview

In essence, in order to amplify any DNA sequence in vitro, the sequence in question must be linked to primary sequence elements capable of directing the replication and propagation of themselves and the linked sequence in the desired target host. The required sequence elements differ according to host, but invariably include an origin of replication, and a selectable marker. 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 that are useful in downstream applications.

Restriction/Ligation Cloning

In the classical restriction and ligation cloning protocols, 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 at various point depending on the particular application; these are summarised as a ‘cloning strategy’.

Isolation of insert

Initially, the DNA fragment to be cloned needs to be isolated. Preparation of DNA fragments for cloning can be accomplished in a number of alternative ways. Insert preparation 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. Chemically synthesised oligonucleotides can also be used if the target sequence size does not exceed the limit of chemical synthesis.Isolation of insert can be done by using shotgun cloning,c-DNA clones,gene machines(artificial chemical synthesis).

Ligation

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 exhibiting DNA ligase activity. 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. Alternatively 3' A overhangs produced by non-proofreading DNA polymerases utilised in PCR can be used. ‘Sticky ends’ allow for both higher efficiency transformations and can be used directional insertion of the insert into the vector, thus minimising the need subsequent screening

Transfection

Following ligation, a portion of the ligation reaction, including vector with insert in the desired orientation 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 used when extremely high transformation efficiencies are required, as in very inefficient cloning strategies. Biolistics are mainly utilised in plant cell transformations, where the cell wall is a major obstacle in DNA uptake by cells.

Selection

Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low efficiency, there is a need to identify the cells that contain the desired insert at the appropriate orientation and isolate these from those not successfully transformed. Modern cloning vectors include selectable markers (most frequently antibiotic resistance markers) that allow only cells in which the vector, but not necessarily the insert, has been transfected to grow.Additionally, the cloning vectors may contain colour selection markers which provide blue/white screening (via α-factor complementation) on X-gal medium. Nevertheless, these selection steps do not absolutely guarantee that the DNA insert is present in the cells. 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.

See also

• Subcloning