Restriction digestion or restriction digest is a molecular biology protocol used to prepare DNA for cloning or analysis. The phenomenon of restriction digestion is seen as DNA fragmentation or the creation of smaller DNA fragments from a larger original DNA molecule.
Restriction digestion employs the function of one or more restriction enzyme to selectively cut DNA strands into shorter restriction fragments. There are many types of restriction digestion cuts that can be generated.
As mentioned there are many types of restriction digestion cuts, and the creation of the final DNA fragment product and end depends on the type of restriction enzyme(s) used.
Blunt-End Restriction Fragments
Blunt end restriction fragments are the result of restriction digestion which yields "blunt" or "non-sticky" end DNA fragments. Non-sticky ends of restriction fragments means the fragments due not have overhangs.
Blunt-end restriction digestion allows the cloning of otherwise incompatible DNA fragments. Blunt-end DNA fragments can also be generated by Klenow polymerase end filling.
Each specific restriction enzyme cuts DNA segments within a specific nucleotide sequence, and almost always breaks the DNA at recognized DNA sequences (the restriction enzyme recognition sequence) to produce the same ends every time.
These recognition sequences are usually 7 nucleotides long however they can range from four to twelve nucleotides long.
As there are four nucleotides - A,C,G and T - exact DNA sequences of four or eight or twelve nucleotides are only found sparcely throughout the genome or a given DNA seqeunce.
Hundreds of restriction enzymes have been isolated from bacteria that are able to digest hundreds of specific sequences.
As a result of this, one can obtain restriction enzymes that can cut most DNA fragments in the desired way.
On the other hand, if the desired restriction sites are not present, they can be inserted into the DNA seqeunce using PCR or other methods.
Once the cuts are made with the restriction enzymes, the plasmid can then be loaded into a gel for electrophoresis. In electrophoresis, the negatively charged DNA is pulled through an agarose gel, causing the shortest fragments of the plasmid to extend farther into the gel, and separating each cut segment from the other. A marker is also loaded with the gel, indicating the amount of base pairs in each segment of the plasmid. The new DNA fragment may then be extracted from the gel by cutting it, and doing a gel purification. Through ligation of a vector which has also been cut by a restriction enzyme, the fragment of interest can then be inserted into this vector, and a new synthetic plasmid is formed. The new plasmid is then transformed into a biological system, and replication occurs.
Restriction Digestion Method
Restriction enzyme digestions are conducted by incubated DNA with restriction enzymes, in appropriate buffers and optimal temperatures for the enzyme.
The amount of salt varies for each restriction enzyme and thus different buffers are used for different enzymes (i.e NEB's #1, #2, #3, and #4.).
When you are conducting double digestions with restriction enzymes, you must take care to use a buffer that is compatible for both enzymes. There are charts supplied by companies such as NEB (look inside their catalog) which help you choose the right buffer.
Restriction Enzyme Digestion Protocol
Usually a normal DNA digestion with restriction enzymes is in a total volume of 50 ul (microliters.)
However, for analytical reactions (to check whether your insert is there etc.) you can do the digest in less volume.
If you want to run your DNA on a "minigel" (30ml agarose gel) you can then digest 0.25 to 2 ug (micrograms) per digest. This is because you can visualize approximately .05ug (50 ng, nanograms) per band on a gel, however this depends on the length of the DNA.
The longer the piece of DNA or band, the easier it is to see.
Sometimes you may need to add (magnesium) Mg++ to the reaction (in the form of MgCl) because you add a large volume of DNA. That is, if "X" is large > 20ul, you need to add Mg.
This is due to the fact that DNA is usually stored in a buffer with EDTA. EDTA bind 4 moles of Mg for every mole of EDTA. Thus, DNA in a 1 mM (milimolar) solution of EDTA will bind 4mM of Mg. A good rule of thumb is for every 20ul of DNA add 1ul of 10mM MgCl.
Note: Enzymes should always be kept cold. Always keep them on ice, and minimize the time you have them out of the Freezer.
Gently pipet to mix and incubate the reaction at 37 C for 1 to 2 hours.
You can inactivate the enzyme by heating at 65 C for some enzymes and 85 C for more tougher enzymes.