Restriction enzymes recognize specific DNA sequences and cut them in a predictable manner.

These enzymes (a.k.a. restriction endonucleases) are part of the genetic engineering toolbox and make gene cloning possible. Naturally, they are defense systems of bacteria against foreign DNA. As they are endonucleases, they can cut foreign DNA from the inside and make it ineffective. 

What makes restriction enzymes suitable for gene cloning is that each of them has a specific restriction site. This is a short DNA sequence (generally 4-8 nucleotide pairs) that an enzyme specifically recognizes. Most restriction enzymes recognize palindromic sequences, meaning that both strands of DNA will have the same sequence when read 5′ to 3′. For example, the sequence ATTGCAAT is palindromic. As soon as this recognition occurs, enzyme cuts the sugar-phosphate backbone from specific points, which are generally within the restriction site. As one would expect, a long DNA molecule naturally has many such restriction sites. Therefore, its digestion with a restriction enzyme generates many smaller DNA fragments – restriction fragments.   

Many restriction endonucleases make staggered cuts in the two strands of DNA, such that the cut ends have a short single-stranded overhang. Because these overhangs are capable of annealing with complementary overhangs, they are called “sticky ends.” Sticky ends facilitate the production of recombinant DNA.

Let’s say we would like to insert our gene of interest into a vector plasmid. As we cut both the DNA fragment containing our gene and the plasmid with the same enzyme, sticky ends form in each of them. These sticky ends can make hydrogen bonds (or anneal) as they are complementary. Another enzyme in the toolbox, DNA ligase, joins these two annealed DNA fragments permanently.  

Practice Questions

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Key Points

• Restriction enzymes are essential tools in genetic engineering; they are in routine use for gene cloning. 

• In nature, restriction enzymes help bacteria to ward off invasion by foreign DNA. 

• Each restriction enzyme recognize a specific, short DNA sequence, a restriction site, and cuts each DNA strand from specific points. 

Key Terms

recombinant DNA: single DNA molecule that is formed by combining DNA molecules from at least two different origins

plasmid: a circular DNA in bacteria and protozoa that is separate from the chromosomes

sugar-phosphate backbone: the outer support of the ladder, forming strong covalent bonds between monomers of DNA.

gene cloning: a biological technique that creates many identical copies of gene (or any other DNA fragment) and directs its replication within a host organism

restriction endonucleases: commercially available enzymes that can target and cut a specific region of DNA; they are naturally found in bacteria

restriction sites: specific sequences of nucleotides, which are recognized by restriction enzymes.

palindromic sequences: nucleic acid sequences where the one strand matches its complementary strand when read in the same direction

restriction fragment: is a DNA fragment resulting from the cutting of a DNA strand by a restriction enzyme

annealing: recombine (DNA) in the double-stranded form following separation by heat

sticky end: produced when the restriction enzyme cuts at one end of the sequence, between two bases on the same strand, then cuts on the opposite end of the complementary strand

DNA ligase: joins DNA strands together