Restriction digestion is a molecular biology technique that utilizes endonucleases, a type of restriction enzyme, to cut DNA at specific sequences, preparing it for other experiments. Endonucleases are essentially molecular scissors with special patterns on their blades that can cut different shapes into DNA. More specifically, endonucleases hydrolyze the phosphodiester bond between sugar and phosphate molecules.

            In the 1960s, Werner Arber was studying “host-controlled restriction of bacteriophages,” a process that prevents bacteriophages from injecting their foreign DNA into a host bacterium. When bacteriophages inject their DNA, the host cell produces restriction enzymes that recognize the foreign DNA and break it down, effectively protecting itself from foreign genetic material. Arber found that this phenomenon consisted of two parts: restriction and modification. Restriction is the breakdown of DNA. Modification involves changing the DNA through processes like methylation to prevent restriction. Hamilton Smith later confirmed Arber’s hypothesis and discovered the first restriction enzyme, HindII, in the bacterium Haemophilus influenzae. Werner Arber and Hamilton Smith, along with microbiologist Daniel Nathans, later received the Nobel Prize in Physiology or Medicine in 1978 for discovering restriction enzymes and their applications to molecular biology.

Restriction digests were first commonly used in medicine to help doctors diagnose diseases caused by genetic mutations. DNA would be collected from patients and cut with specific endonucleases at known sites. The resulting DNA fragments would then be compared against patients without the suspected disease, allowing doctors to get an idea of where a genetic mutation could have occurred. This way of diagnosing genetic diseases has now been replaced with genetic sequencing and screening techniques, but restriction digests are still commonly used in research.

At Sanford Research, Anthony Restaino, an MD/PhD student in the Vermeer Lab, used restriction digests in his PhD work studying peripheral sensory nerves in head and neck cancer. Restaino states that restriction digests are commonly used to introduce new, specific pieces of DNA into circular DNA called plasmids. Endonucleases, which “essentially cleave DNA at set patterns,” then create “overhanging tabs,” says Restaino. These “overhanging tabs” match up perfectly with the new DNA segments scientists want to introduce. These new mutation-containing plasmids can then be added to cells.

            Although it may initially seem intimidating, Restaino says that restriction digests are an extremely useful tool for biologists. “I think what is still very beneficial is that this is a very well-established technique in biology. It is utilized widely by all labs,” says Restaino. “Everyone at some point has probably done it, which makes the universality of it much more approachable.”

Restriction Digest Steps:

1. All reagents are collected and placed on ice to prevent them from degrading. Reagents including your DNA, buffer, and restriction enzymes are pipetted into PCR tubes.
2. The reaction is usually incubated for an hour at digestion temperature (~37℃).
3. After the incubation period, the reaction is stopped using heat or by adding EDTA to the reaction tube.
4. The digested DNA can now be used for other experiments.

            Restaino states that there are many benefits to performing restriction digests. Compared to other gene editing methods, such as CRISPR, restriction enzymes are relatively inexpensive. Restaino explains that “there's sometimes size restrictions for the things that you can put into CRISPR. You don’t necessarily have that same size restriction with a [plasmid].”

            Restriction digestion is a useful technique for cutting DNA to prepare it for analysis or other experiments. Restriction digests are affordable, yet effective and trusted methods used throughout most research labs.