Molecular cloning—not to be confused with cell cloning or whole animal cloning—is an important tool in our endeavour to understand the structure, function and regulation of individual genes and their products. An important resource is the creation of a DNA library, which is a complete collection of DNA fragments, each located singly within a cloning vector that is capable of replication when transferred to an appropriate host cell. Two distinct types of library can be formed, depending on whether the cloned DNA was derived by fragmentation of the genome (genomic library) or was copied from cellular messenger RNA (cDNA library). The following basic steps are involved in the molecular cloning of genes.
a. Choice of DNA
The first step involves choosing which DNA to clone, genomic DNA or cDNA? If the cloned DNA is to be representative of the entire genome of a particular species (ie. genomic DNA - facilitating genome analysis, the control of gene expression, gene structure and intronic sequences), total DNA must be prepared from somatic cells of that species.
Because genomic DNA is identical throughout all somatic cells of an individual, the exact cellular source of material is not important. However, if the DNA is to be representative only of the coding regions of those genes expressing proteins, complementary DNA (ie. cDNA - representing the mRNA population currently active in the cell and indicative of proteins being actively synthesized) must first be created.
cDNA is so-called because it is complementary to cellular mRNA. Because many genes are switched off in different cells at different times, the identities and amounts of transcribed mRNA are in constant flux within a cell, varying at different stages of development and in response to the changing needs of the cell. Consequently, it is important that the tissue or cell type is carefully chosen with respect to age, function and stage of the cell cycle, prior to cDNA production.
b. Insertion of the chosen DNA into a vector
The cloning of DNA requires the production of large quantities of the DNA of interest. This is accomplished by taking advantage of the fact that bacteria and bacteriophage replicate their DNA with high fidelity, many times, in a relatively short time-frame.
There are two sorts of cloning vector: viral vectors (known as bacteriophage, or simply, phage) and plasmid vectors.
Transformation
Typically, phage transformation is used when substantial replication of material is required, being up to 200-fold more efficient than plasmid vectors, the latter being used when the final step involves the preparation of pure DNA. Many contemporary vectors incorporate features from both phage and plasmids and are known as phagemid vectors.
The animation below encapsulates the processes involved in either plasmid or phage transformation. Play the animation to see a schematic of vector formation using a restriction enzyme. A recombinant vector DNA molecule containing foreign insert DNA is formed.
