Restriction enzyme cloning, or “restriction cloning” for short, makes use of DNA restriction enzymes to cut a vector and an insert at specific locations so that they can be easily joined together by the enzyme DNA ligase.
The History of Restriction Enzyme Cloning
“Cloning” as a general laboratory procedure involves making multiple copies of a DNA fragment to enable it to be more easily studied and manipulated.
Most commonly cloning is achieved by inserting one or more DNA fragments of interest, often referred to as “inserts”, into a circular self-replicating DNA called a plasmid vector. When the joined plasmid/insert is introduced into a bacterium the circular plasmid/insert replicates to create hundreds of identical “cloned” copies per cell.
Prior to the 1970s, scientists were not able to easily isolate and study individual genes from organisms. The first advance needed was the discovery of restriction enzymes and the DNA ligase enzyme. This key discovery coupled with the description of scientific protocols enabled scientists to use these tools to isolate individual genes from a genome.
The second major advance in the field was the development of plasmid cloning vectors which could be used to receive and replicate these isolated pieces of DNA. The development of these tools led to the publication of the first recombinant DNA molecules in 1972.
Today, restriction cloning is one of the most commonly used cloning methods. In fact, more than 70% of all molecular biology experiments begin with the restriction cloning of DNA fragments.
How do you select a suitable host for cloning?
Most cloning experiments are performed in a laboratory strain of the bacteria E. coli. Several strains have been developed over the years that emphasize the bacteria’s ability to be a host to a variety of vectors.
Typically this means that the methylation and restriction systems that are native to the bacteria are either deleted or mutated to become non-functional. This ensures that any foreign DNA is not modified after being transformed into the bacteria.
What are the characteristics of a vector system?
In addition to selecting a suitable host, you must also choose a suitable vector. Quality vectors typically have four characteristics:
- An origin of replication enabling the vector to replicate and be maintained within the host cell.
- A multiple cloning site that enables you to insert your DNA into the vector in a standard, unique location.
- A selection marker enabling only bacteria that have taken up the vector to grow and survive on selective growth media. Most commonly, the selection is achieved by the inclusion of an antibiotic resistance gene on the plasmid vector.
- A cloning selection marker that signals the vector contains inserted DNA.