Since its discovery, restriction enzymes have played a major role in the advancement of many important biotech experiments. Given its importance, here’s a quick guide with everything you need to know about restriction enzymes and the cool experiments you can do with them!
Also known as restriction endonucleases, they are a class of enzymes that are naturally occurring in bacteria and provide protection from viral DNA. About 3,000 restriction enzymes have been discovered. Restriction enzymes act like molecular scissors by recognizing specific sequences of DNA and cutting double stranded DNA at these sites. The cutting leaves small fragments of DNA, which can then be utilized in different types of experiments.
The restriction enzyme recognition sites are double stranded palindromic sequences, meaning that the sites contain symmetrical sequences. These sites are highly specific and are usually 4 to 8 base pairs in length. Near, or around this site, the restriction enzyme will cut the phosphate backbone of the double stranded DNA, resulting in DNA fragments. Different restriction enzymes produce different types of fragments depending on its recognition sites and the position of the cuts. The DNA fragments can result in two different types of ends: sticky ends or blunt ends. Sticky ends are produced from staggered cuts, where the restriction enzyme cuts at different positions resulting in complementary overhangs. If the DNA is cut at the same position, the DNA fragments will have blunt ends.
A piece of DNA can be cut several times by a restriction enzyme. Depending on the length of the recognition site (n), the DNA will be cut 4n times. Therefore, the probability that a piece of DNA will be cut is higher the longer the piece of DNA is. Depending on the position of the recognition site, the cuts can produce a variety of fragments that are of varying lengths.
The Role of Restriction Enzymes in Experiments
In molecular cloning, restriction enzymes will cut plasmid DNA at multiple recognition sites, resulting in a variety of differently sized fragments. These fragments are run on an agarose gel so that the size of each fragment can be determined. Using this information, a plasmid map can be created showing the position of each restriction enzyme site and the distance that they are from each other.
In DNA fingerprinting, restriction enzymes will cut DNA into differently sized fragments. These fragments are run on an agarose gel to determine the size of each fragment. Analyzing the restriction enzyme pattern of suspect DNA samples in comparison to crime scene DNA samples can result in identifying the correct suspect.
In genotyping with polymerase chain reaction (PCR), restriction enzymes will cut an amplified region of DNA produced by PCR from a particular gene. Analyzing the restriction enzyme pattern of the amplified region of DNA will determine a person’s genotype of that region of DNA.
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