Restriction enzymes, also known as restriction endonucleases, function as molecular scissors within cells, specifically cutting DNA molecules. Each restriction enzyme recognizes and binds to a unique sequence of DNA, referred to as its restriction site. These enzymes are naturally found in bacteria, where they serve as a defense mechanism against invading viruses by cleaving foreign DNA.
BamHI is a widely used example of a Type II restriction enzyme, a class of enzymes that cut DNA at specific nucleotide sequences. It was originally isolated from the bacterium Bacillus amyloliquefaciens H. This enzyme is widely used in molecular biology laboratories for manipulating DNA sequences due to its precise cutting ability.
The BamHI Recognition Sequence
The specific DNA sequence that BamHI recognizes and cuts is 5′-GGATCC-3′. This sequence is palindromic, meaning it reads the same forwards and backwards on both complementary strands of the DNA molecule. The complementary strand would read 3′-CCTAGG-5′.
BamHI makes a staggered cut within this recognition sequence, cleaving the DNA backbone after the 5′-guanine on each strand. The result of this staggered cleavage is the creation of a 4-base single-stranded overhang, commonly known as a “sticky end” or cohesive end. For BamHI, this sticky end has the sequence 5′-GATC-3′. These overhanging ends can easily base-pair with other DNA fragments cut with the same enzyme, due to their complementary nature.
Function in Creating Recombinant DNA
The ability of BamHI to create specific sticky ends makes it particularly useful in the process of molecular cloning, which involves combining DNA from different sources. In this process, BamHI is used to cut both a target gene, often called the “insert,” and a circular DNA molecule known as a plasmid, which serves as a “vector” to carry the gene.
When both the insert DNA and the plasmid vector are cut with the same restriction enzyme, such as BamHI, they both acquire identical, complementary sticky ends. These complementary sticky ends can then spontaneously anneal, or base-pair, with each other when mixed together.
Once the sticky ends have annealed, another enzyme, DNA ligase, is introduced into the reaction. DNA ligase forms new phosphodiester bonds, which are the stable chemical links in the DNA backbone, thereby permanently joining the insert DNA into the plasmid vector. This sealing process creates a new, hybrid DNA molecule known as “recombinant DNA,” which can then be introduced into a host cell for replication or expression.
Experimental Factors and Star Activity
For BamHI, like other restriction enzymes, to function correctly, specific experimental conditions are maintained. This includes using a particular buffer, which helps maintain an optimal pH and a suitable salt concentration. The reaction is also carried out at 37°C, the optimal temperature for the enzyme’s activity.
Under certain non-optimal conditions, restriction enzymes can exhibit what is known as “star activity”. This phenomenon occurs when the enzyme loses its specificity and begins to cleave DNA sequences that are similar but not identical to its precise recognition site.
Star activity can lead to unintended cuts in the DNA, which can compromise the accuracy and success of molecular cloning experiments. Several factors can induce star activity, including high concentrations of glycerol in the reaction mixture. Other contributing factors include a high ratio of enzyme units to the amount of DNA, low ionic strength in the buffer, or the presence of organic solvents like ethanol or DMSO. To avoid star activity, researchers carefully control these conditions, ensuring proper buffer composition, and minimizing contaminants.