Gene cloning

The basis of gene cloning technology owes its origins to the realisation that two different practical techniques (the use of restriction enzymes and the use of bacterial plasmids) could be utilised to generate chimeric DNA molecules in large quantities. Restriction enzymes are endonucleases that recognise and cut DNA at specific sequences within the molecule (Figure 14.1a). They recognise short four- or six-base pair sequences and either cut at opposite ends of these sequences, generating overlapping ends known as ‘sticky’ ends because they can reanneal using allowed base pair rules, or they cut both strands at the same site to generate ‘blunt’ ends.

Plasmids are small circular DNA molecules that can be present in a bacterial cell and replicate along with the bacterial genome (Figure 14.1b). They can code for proteins such as antibiotic-resistance proteins (Figure 14.2) and are responsible for creating many multi-drug-resistant bacteria.

The combination of these two technologies led to the production of recombinant DNA molecules. If a plasmid preparation is cut with a particular restriction enzyme, small lengths of DNA with identical sticky ends will form. Similarly, if a piece of human DNA is cut with the same restriction enzyme, short sequences of DNA will result with the same sticky ends. The two sets of DNA pieces can be incubated together and allowed to reanneal. The annealed DNA pieces can then be covalently joined in vitro using DNA ligase. The result will be a mixture of reformed plasmid molecules in addition to some plasmids into which human DNA sequences have been inserted. It is now possible to use selection techniques to select for only the chimeric human/plasmid molecules. These chimeric DNA molecules will be replicated by the host bacteria. The plasmid acts as the vector, to produce large quantities of the human molecules. The human DNA sequences can then be extracted using the same restriction enzyme that was used to cut the DNA initially.

Libraries

If a sample of total human DNA is treated with a restriction enzyme and the fragments are inserted into a vector, the chimeric molecules generated will contain the sum total of all the DNA sequences present in the human genome. The product of this process has been called a human genomic library. It can be prepared from the DNA of human tissue or human cell lines and will not be dependent on the source of DNA. Much of our understanding of the sequences in human genes has come from studies on human DNA libraries. However, these sequences cannot be used to generate human proteins in a bacterial protein synthesising system because bacterial cells cannot process the complex intron/exon structure of human genes.

If the total mRNA is isolated from a specific human tissue or cell line it can be reverse transcribed into complementary DNA in vitro using the RNA virus enzyme reverse transcriptase. The complementary DNA sequences can be ligated into a particular vector. The vector will then contain DNA copies of all the mRNA molecules present in that tissue. This is known as a complementary DNA (or cDNA) library. Unlike the genomic library, a cDNA library will be specific to the tissue used to isolate the mRNA because different tissues express different genes at different times. cDNA libraries can be used to produce large quantities of proteins.