Edvotek Instructional Video: All About Restriction Enzymes

Restriction endonucleases (also known as restriction enzymes) act like molecular scissors, cutting double-stranded DNA at specific sequences. They are produced by many species of bacteria to protect themselves from invading viral DNA. The utility of restriction enzymes has made molecular cloning, DNA mapping, sequencing and various genome-wide studies possible, launching the era of biotechnology.

Learn more about restriction enzymes in this short video, or with the below resources:

Edvotek Hands-on Restriction Enzyme Experiments: https://www.edvotek.com/search?keywor...
Biotech Basics: Restriction Enzymes: https://blog.edvotek.com/2014/06/13/b...
In the Spotlight: EcoRI, HindIII, and BamHI: https://blog.edvotek.com/2023/02/21/i...
Edvotek at Home: Restriction Enzyme Analysis of DNA: https://blog.edvotek.com/2020/03/26/e...
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Restriction endonucleases are one of the most significant discoveries of molecular biology. These enzymes can cut DNA in a specific and reproducible manner.

Restriction endonucleases evolved in bacteria as a defense against viral attacks. When viral DNA enters the bacterial cell, these enzymes recognize foreign DNA based on specific modifications, and chop it into smaller pieces to prevent replication.

The first restriction enzymes were purified in the 1970’s using techniques like ion exchange chromatography, which separates proteins based on their charge, or size exclusion chromatography, which separates proteins by their size. Today, affinity chromatography is used to purify restriction enzymes by tagging proteins with specific amino acid sequences that interact with the chromatography matrix.

Type II restriction enzymes are one of five types of endonucleases isolated from bacteria. They bind to DNA as homodimers, with two identical protein subunits binding to each other. Each protein contains a DNA binding domain and an endonuclease domain for cleaving the DNA. Most of these enzymes require magnesium ions as a cofactor, meaning that we must use the correct buffer for best results.

Restriction enzymes can cleave the recognition site at the center of the DNA strands to yield a blunt end or at a staggered position, leaving overhangs called sticky ends. The likelihood that an enzyme will cut a piece of DNA increases proportionally with the length of its recognition site. An enzyme typically makes one cut for every 4 to the n base pairs. Therefore, the longer a DNA molecule is, the greater chance that it contains one or more restriction sites.

Digestion of the same DNA with different enzymes creates unique patterns that can provide information about a gene or a genome without knowing its sequence. This was essential before DNA sequencing became more inexpensive and readily available. In fact, this is the principle behind DNA fingerprinting.

In 1978, the Nobel Prize honored “the discovery of restriction enzymes and their application to problems of molecular genetics.” These enzymes remain an indispensable part of the molecular biotechnology toolbox.