Molecular cloning

Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1]

Overview

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.

Overview

In essence, in order to amplify any DNA sequence in vivo, 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 specialized 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.

Recombinase based Cloning

A novel procedure of cloning or subcloning of any DNA fragment by inserting the special DNA fragment of interest into a special area of target DNA through interchange of the relevant DNA fragments .

This is an one-step reaction: simple, efficient, facilitating high throughput or automatic cloning and/or subcloning .

Restriction/Ligation Cloning

In the classical restriction and ligation cloning protocols, cloning of any DNA fragment essentially involves four steps: DNA fragmentation with restriction endonucleases, ligation of DNA fragments to a vector, transfection, and screening/selection. Although these steps are invariable among cloning procedures a number of alternative routes can be selected at various points depending on the particular application; these are summarized 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 polymerase chain reaction, but it may also be accomplished by restriction enzyme digestion, DNA sonication and fractionation by agarose gel electrophoresis. Chemically synthesized 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).

Transformation

Following ligation, the ligation product (plasmid) is transformed into bacteria for propagation. The bacteria is then plated on selective agar to select for bacteria that has your plasmid of interest. Individual colonies are picked and tested for the wanted insert. Maxiprep can be done to obtain large quantity of the plasmid containing your inserted gene.

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 specialized 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 utilized 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.

Blank Cell Cloning

This form of cloning is fairly simple. A nucleus is extracted from a cell, thus leaving the cell with no genetic instructions. After this step, the nucleus of another cell is injected into the cell where the nucleus was removed. The cell receives an electric shock, thus forcing it back to life. The cell can then be forced into rapidly reproducing, thus producing new genetic material.

References

• Copeland NG, Jenkins NA, Court DL. Recombineering: a powerful new tool for mouse functional genomics.

Nat Rev Genet. 2001 Oct;2(10):769-79. Review. PMID: 11584293

• Lu JP, Beatty LK, Pinthus JH. Dual expression recombinase based (DERB) single vector system for high throughput screening and verification of protein interactions in living cells.". Nature Precedings 2008 <http://hdl.handle.net/10101/npre.2008.1550.2>blablabla.

See also

• Cloning
• Subcloning
• Polymerase chain reaction