In its simplest form, for example in the treatment of single gene (monogenic) disorders, gene therapy can be considered the replacement of a defective gene in an individual with a functional “wild type” version of the gene to reverse the pathology. Cloning genes can be useful in curing and treating genetic disorders such as cystic fibrosis and severe combined immunodeficiency (SCID).
The initial stage of cloning a gene is to generate a DNA fragment containing the gene of interest to be cloned. These fragments can be produced using restriction endonucleases or by PCR [to introduce convenient restriction sites when they are lacking from the fragment of interest].
Restriction endonucleases were first discovered in the 1960s and are enzymes originally isolated from bacteria that evolved to cut up viral DNA and to prevent bacteria from being invaded by viruses that inject their DNA into them in order to take over the cell. There are many types of restriction endonucleases which all cut DNA at specific recognition sequences (http://www.neb.com/tools-and-resources/selection-charts/alphabetized-list-of-recognition-specificities) and their discovery and use has revolutionised gene cloning since the 1990s.
Certain types of restriction endonucleases cut DNA in a staggered manner and, therefore, leave each strand of DNA with a ‘sticky end’- a single strand of DNA only a few nucleotide bases long that overhangs from the cut site. ‘Sticky ends’ are important for cloning DNA because two pieces of DNA cut with the same restriction endonuclease will have ‘sticky ends’ that are complementary to one another and can be joined together using the enzyme DNA ligase. It is possible to combine the DNA of one organism with that of any other if they have been “cut” using the same restriction endonuclease, then “pasted” back together using a DNA ligase.
The next stage, after the DNA fragments have been produced, is to join the fragments to a vector of which the most commonly used is the plasmid (“ligation”). The same (or compatible) restriction endonuclease that is used to produce the DNA fragments is used to cut open the plasmid to ensure that the ‘sticky ends’ will be complementary to each other. When the open plasmids are mixed with the DNA fragments the ‘sticky ends’ can interact with each other and a ligation enzyme (DNA ligase) can re-anneal the fragments together permanently.
Subsequently, the recombinant plasmid must be reintroduced into bacterial cells in a process called transformation. In this process the plasmid is mixed with specially prepared bacterial cells in a medium containing calcium ions. This preparation is heated in a water bath (to “heat shock” the bacteria and make them more amenable to taking up the plasmid) and then cooled on ice. The calcium ions together with the changes in temperature cause the bacteria to become permeable thus allowing the plasmids to enter into the cytoplasm through the bacterial cell membrane. Only a small proportion of bacteria will take in the plasmids and thus many will not possess the DNA fragments. To prevent plasmids re-ligating back together without taking up the relevant insert, the linearised plasmid backbone is treated with an enzyme called SAP (shrimp alkaline phosphotase) which removes phosphate groups from the cleaved ends and makes them less “sticky”. This is important as it: (1) prevents the plasmid backbone from re-circularising, and (2) increases the likelihood that the plasmid backbone will accept the alternative DNA fragment which has not been de-phosphorylated.
To distinguish between the bacterial cells which have taken in the plasmid and those that have not (selection), the bacteria are grown in a medium containing an antibiotic. Because the transformed bacteria (which have taken up the plasmid DNA) contain the genes for the resistance of this antibiotic they are able to grow in the media containing the antibiotic whilst all the bacteria which were not successfully transformed will die. The plasmids can be extracted from the bacteria and a number of methods, such as DNA sequencing, can be used to identify plasmids that have incorporated the cloned gene. DNA sequencing is crucial to ensure that the correct DNA sequence has been inserted and in the correct orientation.
Once the bacterial cells which contain the plasmid with the cloned gene have been isolated and identified, the bacteria can be used to make many copies of the plasmid for medical or commercial use. This method of gene cloning has been vital to the success of gene therapy and medical research in general.