DNA cloning is a basic tool which allows researchers to produce multiple copies of a specific section of a nucleic acid, typically a single gene or set of genes.
When this process is carried out, it usually involves the insertion of a gene into a plasmid—a circular piece of DNA found in the cytoplasm of bacterial cells which is regulated independently of the cell’s chromosomal DNA. When used in DNA cloning, the plasmid is also known as a vector.
The gene of interest is inserted into the plasmid by means of bacterial enzymes called restriction enzymes, which cut DNA at specific base pair locations. To do this,
the plasmid, gene fragment, and enzymes are all incubated together. The enzymes cut the DNA sections at specific locations, leaving a mixture of gene fragments and plasmids, each with compatible base pair sequences at their ends. This mixture is then incubated with a second enzyme called DNA ligase, which joins the fragments and plasmids together to form a new plasmid with the gene of interest inserted into it.
The resulting plasmid is called a recombinant DNA molecule, because it has DNA from two different sources recombined into a new single molecule.
The next step is inducing bacteria to take up the recombinant DNA molecules. Bacteria do this naturally via a process called transformation, which typically occurs between two bacterial cells which share plasmids (this is one way in which genes causing antibiotic resistance can be shared among bacterial cells in a population).
However, there is a problem with this procedure: some bacteria will take up the recombinant DNA, but others might take up non-recombinant DNA. How is it possible to determine which is which?
The key is using a plasmid vector which contains what is called a marker. Typically this is an antibiotic resistance gene. Then, the bacteria can be grown on medium which contains the antibiotic—only those bacteria which have taken up the recombinant plasmid will be able to grow on the medium.
Once the bacteria have been plated on medium containing the antibiotic, all the cells which grow will contain the selective marker. However, it is still necessary to identify a colony which actually contains the gene of interest.
To do this, a technique called nucleic acid hybridization is often used. This technique works by base-pairing the gene of interest with a probe—a nucleic acid molecule which has base-pairs complementary to a sequence in the gene. The probe is a single-stranded DNA molecule which has a radioactive marker attached, to allow for identification.
When the probe and the gene
are allowed to mix, they are able to bind together, forming a double-stranded DNA molecule, which can be identified due to its radioactivity. Any bacterial colony containing DNA which binds to the probe, therefore, contains a copy of the gene of interest. The colony which contains the DNA is said to be a clone.