Title: Restriction Enzymes
Course: BTEC-1100-301-302-303-F21 > Section: Module 4 - Applied Molecular Biology

Restriction Enzymes and Ligases

In recombinant DNA technology, DNA molecules are manipulated using naturally occurring enzymes derived mainly from bacteria and viruses. The creation of recombinant DNA molecules is possible due to the use of naturally occurring restriction endonucleases (restriction enzymes), bacterial enzymes produced as a protection mechanism to cut and destroy foreign cytoplasmic DNA that is most commonly a result of bacteriophage infection. Stewart Linn and Werner Arber discovered restriction enzymes in their 1960s studies of how E. coli limits bacteriophage replication on infection. Today, we use restriction enzymes extensively for cutting DNA fragments that can then be spliced into another DNA molecule to form recombinant molecules. Each restriction enzyme cuts DNA at a characteristic recognition site, a specific, usually palindromic, DNA sequence typically between four to six base pairs in length. A palindrome is a sequence of letters that reads the same forward as backward. (The word “level” is an example of a palindrome.) Palindromic DNA sequences contain the same base sequences in the 5ʹ to 3ʹ direction on one strand as in the 5ʹ to 3ʹ direction on the complementary strand. A restriction enzyme recognizes the DNA palindrome and cuts each backbone at identical positions in the palindrome. Some restriction enzymes cut to produce molecules that have complementary overhangs (sticky ends) while others cut without generating such overhangs, instead producing blunt ends.

Molecules with complementary sticky ends can easily anneal, or form hydrogen bonds between complementary bases, at their sticky ends. The annealing step allows hybridization of the single-stranded overhangs. Hybridization refers to the joining together of two complementary single strands of DNA. Blunt ends can also attach together, but less efficiently than sticky ends due to the lack of complementary overhangs facilitating the process. In either case, ligation by DNA ligase can then rejoin the two sugar-phosphate backbones of the DNA through covalent bonding, making the molecule a continuous double strand. In 1972, Paul Berg, a Stanford biochemist, was the first to produce a recombinant DNA molecule using this technique, combining the SV40 monkey virus with E. coli bacteriophage lambda to create a hybrid.

(a) In this six-nucleotide restriction enzyme site, recognized by the enzyme BamHI, notice that the sequence reads the same in the 5ʹ to 3ʹ direction on both strands. This is known as a palindrome. The cutting of the DNA by the restriction enzyme at the sites (indicated by the black arrows) produces DNA fragments with sticky ends. Another piece of DNA cut with the same restriction enzymecould attach to one of these sticky ends, forming a recombinant DNA molecule. (b) This four-nucleotide recognition site also exhibits a palindromic sequence. The cutting of the DNA by the restriction enzyme HaeIII at the indicated sites produces DNA fragments with blunt ends. Any other piece of blunt DNA could attach to one of the blunt ends produced, forming a recombinant DNA molecule.

Here is a video of what it may look like for a restriction enzyme to cleave DNA and for ligase to the ligate another piece of DNA into that cleavage site.

Restriction Enzyme EcoR1

You will be cleaving your bglA-pLATE31 recombinant plasmid with two restrictions enzymes to verify that you have successfully cloned the bglA gene.  Given the following pLATE31 vector map and knowing that the bglA gene is 1344 base pairs in length, what size fragments do you predict will be generated by a restriction digest using both XbaI and StuI enzymes?