Molecules of DNA can be hundreds of thousands of basepairs long, so to efficiently work with them, it is oftentimes necessary to cut the DNA into smaller, more manageable lengths. This cutting of DNA is called digestion, as it is carried out using enzymes. Known as restriction endonucleases, these enzymes are highly specialized and digest DNA at a specific sequence of nucleotides. This stretch of DNA is known as a recognition sequence. When the enzyme encounters this sequence, it hydrolyzes the phosphodiester backbone of the DNA. The exact point within the recognition sequence at which the phosophodiester backbone is cleaved is known as the restriction site. Each enzyme recognizes a specific recognition sequence and this inherent specificity allows for exquisitely fine-tuning of DNA products. While each enzyme recognizes a specific sequence, it is possible for multiple enzymes to recognize the same site. Different enzymes that recognize the same site are called isoschizomers.
Currently, over 4000 restriction endonucleases have been identified. Of these, greater than 600 are commercially available for laboratory use. Restriction endonucleases can be further delineated by their cut position, cofactor requirements, and modification preference: Types I, II, III, and IV. Type II enzymes are the most commonly-used for molecular biology, as they recognize the canonical 4-8 nucleotide sequence, cut at a reliable position within that site, and requires only magnesium as a cofactor. Unlike other restriction enzymes, Type IV enzymes preferentially cleave methylated DNA and have become useful as tools for epigenetics.