> See also: > - Reference # Restriction Enzymes **Restriction enzymes** are *nucleases* that recognizes a specific, short nucleotide sequence of DNA (known as a restriction/cut/target site) and then cuts the DNA only at that specific site. These were first discovered in bacteria (and other prokaryotes) and functioned as their *innate immune system* against viruses and bacteriophages. Similarly, [[CRISPR]] can be viewed as bacteria’s adaptive immune system. - The bacterial cells are able to tell the difference between viral and self DNA through methylation --- The *cut frequency* of a restriction enzyme depends on the length of its recognition sequence. $\text{Cut Frequency} = \frac{1}{4^n}$ --- ![[Restriction Enzymes Guide.pdf]] You can build restriction maps by analyzing the fragments formed after a restriction enzyme(s) interacts with a sequence. - Similarly to [[Gene Mapping]] ## Cleavage Types There are many different known **restriction enzymes** which can vary in *how they cleave* the **recognition sequence**. - *Sticky Ends (Overhangs)* - - *Blunt Ends* - --- Some unique restriction enzyme variations: - **Isoschizomers:** cut the same sequence at the same location (identical) - **Neoschizomers:** cut the same sequence in different locations, producing different overhangs - If you wanted to guarantee gene directionality when cloning you would likely need to use two restriction enzymes that produce different overhang cut sites. ### Restriction Enzyme Buffers/Prerequisites **Star activity** is when enzymes cut non-specifically under certain conditions - *Factors include:* buffer, pH, ions, ionic strength, high DNA:enzyme ratio, and presence of volume excluders (glycerol/ethylene glycol) - *"High fidelity" enzymes* aim to reduce star activity