Molecular Cloning Outline

Molecular cloning provides a convenient way of breaking very large segments of DNA associated with genomes into smaller pieces that are suited to detailed analysis. Cloning involves the amplification of specific segments of DNA leading to the accumulation of large amounts of identical DNA fragments that can be used for various purposes. Classical molecular cloning employs bespoke DNA cloning vectors, which are often derived from circular DNA molecules called bacterial plasmids and bacteriophage; such recombinant vectors replicate efficiently in specialised strains of bacterial host cells, resulting in the production of multiple copies of the recombinant DNA molecule that was inserted into the vector. Before a DNA fragment can be inserted into a cloning vector, it must be prepared so that the precise DNA sequences of its ends are compatible with the ends of the vector molecule DNA sequence.
This is achieved using commercially available enzymes that cleave double-stranded DNA at specific sequences; these are known as restriction enzymes or restriction endonucleases. In Nature, restriction enzymes constitute part of the bacterial defence system by digesting double-stranded DNA from harmful bacteriophage and other foreign DNA as it enters the cell. To date, more than 3500 restriction enzymes have been identified, some of which are commercially available and used for routine molecular biology applications.If both the vector and the fragments of ‘foreign’ DNA are cut with the same restriction enzyme, such as EcoRI, which cuts at the sequence 50GAATTC30, it is a relatively straightforward procedure to join, or ligate, the two molecules because their matching ends have small overhangs with complementary base pairs (Figure ). The resulting molecule is a recombinant vector which is capable of growing in the host cell.

Figure : Overview of the restriction endonuclease digestion and DNA ligation process in DNA cloning.

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