These single nucleotide polymorphisms, or SNPs, are difficult to detect with PCR and gel electrophoresis alone. Restriction enzymes can also be used in subcloning to isolate a fragment of DNA from one plasmid and insert into another, so the desired fragment can be replicated using bacteria.īy employing the polymerase chain reaction, or PCR to introduce restriction sites into genes at very specific locations, restriction enzymes can be used to determine the presence of single nucleotide differences in alternate forms of the same gene, or allele. The different banding patterns of the same gene from a given species, or in this case different species, are called restriction fragment length polymorphisms. Researchers can examine DNA fragment sizes, produced by the digest, in order to determine the authenticity of fish samples. By loading a digest into a specialized chip and then placing that chip into a machine called a bioanalyzer. Restriction enzymes can be used diagnostically, in order to identify particular samples. Now that we have seen how digests are carried out, lets have a look at various ways restriction enzymes can be used. The use of control DNA with known restriction sites allows the activity of the enzyme to be tested. For example, a no enzyme control will allow you to check the integrity of DNA sample and determine if exonuclease activity is present. Using controls are a good way to understand why a digest might go wrong. Another way to overcome buffer incompatibility and perform a sequential digest is to purify the DNA following the initial digest and then perform the second digest. For example, the digest can be performed with one enzyme first, and then the buffer composition, can be altered in order to be optimal for the second enzyme. Sometimes, however, you’ll find incompatibility in the reaction conditions between the two enzymes, and in this case the workaround is to use the enzymes sequentially. In this case you need to check that buffer conditions and incubation temperatures are compatible between the two enzymes, if so, then you can perform a double digest and have both enzymes cut in the same reaction. Sometimes you may find yourself in a situation where multiple enzymes need to be used to generate a specific DNA fragment. Here are a couple of helpful hints for running your digests and ensuring success. While restriction enzymes cut site-specifically most of the time, prolonged incubation times can lead to star activity, which is cutting at sites that are similar, but distinct from their typical digestion sites.įollowing inactivation, the DNA should be run on an agarose gel to ensure that the digest was successful. Once your digest has completed, it’s a good idea to incubate the reaction mixture at 65˚C to heat inactivate the restriction enzymes. Then incubate at an optimal temperature for your restriction enzyme, usually 37☌ in a heating block for 1 to 4 hours. After, mix by vortexing and then centrifuge briefly at 12,000xg in a microcentrifuge to collect the contents at the bottom of the tube. Units are defined as the amount of enzyme required to produce a complete digest of 1μg of control DNA in 60 minutes at 37☌ in a 50μL reaction volume. Then 10x Restriction Enzyme Buffer, then BSA if needed, up to 1μg of DNA, and 2-10 units of enzyme. First, a volume of sterile, nuclease-free water that will yield a final reaction volume of 20μL. To a microfuge tube reaction components should be added in the following order. Keep the restriction enzyme on ice or a thermal resistant container to make sure there is optimal activity for future reactions. To begin setting up the digest, retrieve the restriction enzyme from the freezer or fridge. Suppliers of restriction enzymes will have resources that one can check to obtain all of the necessary information. Each restriction enzyme can potentially have different buffer conditions, incubation temperatures, and requirements for BSA.
BSA will stabilize the reaction by preventing enzyme from sticking to the side of the container that houses the digest.
A digestion reaction typically consists of the following: deionized water, the DNA that’s going to be cut, buffer specific to the enzyme you will use, and sometimes a protein called bovine serum albumin or BSA. A restriction enzyme digest should be carefully planned.
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