Like all enzymes, a restriction enzyme works by shape-to-shape matching. When it comes into contact with a DNA sequence with a shape that matches a part of the enzyme, called the recognition site, it wraps around the DNA and causes a break in both strands of the DNA molecule.

Restriction enzymes hydrolyze covalent phosphodiester bonds of the DNA to leave either “sticky/cohesive” ends or “blunt” ends. This distinction in cutting is important because an EcoRI sticky end can be used to match up a piece of DNA cut with the same enzyme in order to glue or ligate them back together.

FAQ: Why is my Restriction Enzyme not cutting DNA? If the control DNA is cleaved and the experimental DNA resists cleavage, the two DNAs can be mixed to determine if an inhibitor is present in the experimental sample. If an inhibitor (often salt, EDTA or phenol) is present, the control DNA will not cut after mixing.

To be able to sequence DNA, it is first necessary to cut it into smaller fragments. Many DNA-digesting enzymes (like those in your pancreatic fluid) can do this, but most of them are no use for sequence work because they cut each molecule randomly. This produces a heterogeneous collection of fragments of varying sizes.

5′ overhang- Restriction enzymes that cleave the DNA asymmetrically leave several single stranded bases. If the single-stranded bases end with a 5′ phosphate, the enzyme is said to leave a 5′ overhang. 3′ overhang- Restriction enzymes that cleave the DNA asymmetrically leave single-stranded bases.

The cleavage of two opposite strands of duplex DNA at points near one another.

Blunt-end ligation Blunt end ligation does not involve base-pairing of the protruding ends, so any blunt end may be ligated to another blunt end. Blunt ends may be generated by restriction enzymes such as SmaI and EcoRV.