What are Restriction Enzymes?
Restriction enzmyes recognize short DNA sequences and cleaves double-stranded DNA at or near a specific recognition site. Restriction enzmyes are classified into four types, based on their subunit structure, cofactor requirements and specificity of cleavage.
3,000 different restriction enzymes have been discovered, which recognize over 230 distinct DNA sequences. These enzymes are routinely used for DNA modification around the world and are an indispensable tool in molecular cloning.
The basis for the research of restriction enzymes goes back to the work of Luria and colleagues in the early 1950s . Luria observed that the bacteriophage λ can grow good in one strain of E.coli (e.g. E.coli C), but often poorly in another E.coli strain (e.g. E.coli K). The host cell (E.coli K) was known as the restriction host and appears to have the ability to reduce the biological activity of the phage λ.
The first-time that the term restriction enzyme was mentioned was in the 1960s in the laboratories of Arber and Meselson. They found out that the restriction is caused by an enzymatic cleavage of the phage DNA. The enzyme involved in this process was termed “restriction enzyme” [2, 3]. The restriction enzymes studied by Arber and Meselson were type I restriction enzymes, which cleave DNA at random places away from the recognition site.
In 1970, Smith and colleagues isolated and describe the first type II restriction enzyme, Hind II . Restriction enzymes of type II are much more useful for laboratory work, because they cleave DNA at the site of their recognition sequence. Due to its importance for molecular biology, Smith, Arber and Nathans shared the 1978 Nobel Prize for Medicine and Physiology for their discovery of restriction enzymes and their application to molecular genetics.
All restriction endonucleases recognize a specific DNA sequence. The recognition sequence is usually palindromic or partially palindromic, meaning the base sequence reads the same backwards and forwards. Restriction enzymes can cleave double stranded DNA either at the center of both strands to yield “blunt ends” or at a staggered position leaving overhangs called “sticky ends”.
Different Types of restriction enzymes
Based on the structure, cofactor requirements and specificity of cleavage there are four types of restriction enzymes (Types I, II, III, and IV).
Type I restriction enzymes cleaves the DNA at a random location far away from the recognition sequence. These enzymes require both ATP and S-adenosyl-L-methionine to function.
Type II restriction enzymes cleaves the DNA within or near the recognition sequence. These enzymes do not require ATP and are independent from methylase. The type II enzymes are the most useful restriction endonucleases for the daily laboratory work. All our NIPPON Genetics EUROPE restriction enzymes are from type II.
Type III restriction enzymes cleaves DNA about 20 – 25 base pairs away from the recognition sequence. They require both ATP and S-adenosyl-L-methionine to function.
Type IV restriction enzymes cleaves only modified, typically methylated DNA in contrast to the types I-III, which are usually inhibited by methylation.
A vector and an insert DNA can be cloned by cleaving with two different restriction enzymes, thus generating two different restriction ends. This strategy prevents the vector from being ligated without an insert, resulting in great reduction in self-ligation and increase in cloning efficiency. Most of our restriction enzymes are 100% active in the FastCut Buffer, making double digestion simple. Please look at the Performance Chart to make a double digestion with the four standard buffers.
Activity of Unique FastGene® Buffer
Nippon Genetics provides four standard buffers that maximally support the activity of each restriction enzyme in the buffer provided with the enzyme. However, some restriction endonucleases require unique buffer for maximal activity. Take a look on the following table to select a buffer for double digestion if a restriction enzyme requires unique buffer. Listed are activities (%) of the most commonly used restriction endonucleases in the five unique FastGene® Buffers for EcoR I, BamH l, Acc III, Bal I, and Dpn Il, respectively. A restriction enzyme, if it is active in one of the four standard buffers, is usually active in a unique buffer. Therefore, it is possible to perform double digestion in a particular unique buffer. If digestion efficiency is low due to the suboptimal buffer, increase the amount of restriction endonucleases or incubate for a longer period of time.
 Luria and Human (1952) A nonhereditary, host-induced variation of bacterial viruses. J Bacteriol., 557-569.
 Arber and Linn (1969) DNA modification and restriction. Annu Rev Biochem., 467-500.
 Meselson and Yuan (1968) DNA restriction enzyme from E. coli. Nature, 1110-1114
 Smith and Wilcox (1970) A restriction enzyme from Hemophilus influenzae. I. Purification and general properties. J Mol Biol., 379-391.