Biotech Basics: Restriction Enzymes

What are 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.

How do restriction enzymes know where to cut DNA?

In general, restriction enzymes recognize palindromic stretches of DNA that are 4-8 base pairs in length.  The restriction enzyme cuts through both strands of DNA, creating fragments with one of two types of DNA ends — “blunt” or “sticky” (Fig. 1).

©Edvotek 2014
©Edvotek 2014

Enzymes like HaeIII cleave through both DNA strands at the same position, which generates fragments without an overhang.  These so-called “blunt” ends can be joined with any other blunt end without regard for complementarity.  In contrast, enzymes like EcoRI cut through the DNA strands at staggered positions, creating short overhangs of single-stranded DNA.  Such overhangs are referred to as “sticky” ends because the single-strands can interact with—or stick to—other overhangs with a complementary sequence.

Cleavage of DNA with Restriction Enzymes ©Edvotek 2014
Cleavage of DNA with Restriction Enzymes ©Edvotek 2014

How many times can a restriction enzyme cut a piece of DNA?

The probability that a given enzyme will cut, or “digest”, a piece of DNA is directly proportional to the length of its recognition site.  Statistically, an enzyme will average one cut for every 4n base pairs, where n is the length of the recognition site.  Therefore, the longer a DNA molecule is, the greater the probability is that it contains one or more restriction sites.  For instance, an enzyme that recognizes six base pairs (e.g., EcoRI) will cut once every 4096 (or 46) base pairs. If EcoRI is used to digest both human chromosomal DNA and a plasmid, it will cut the chromosomal DNA over 700,000 times (3 billion base pairs, cut every 4096 base pairs), but may only cut the plasmid once (5,000 base pairs, cut every 4096 base pairs).