A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded or single stranded DNA at specific recognition nucleotide sequences known as restriction sites.[1][2][3] Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses.[4][5] Inside a bacterial host, the restriction enzymes selectively cut up foreign DNA in a process called restriction; host DNA is methylated by a modification enzyme (a methylase) to protect it from the restriction enzyme’s activity. Collectively, these two processes form the restriction modification system.[6] To cut the DNA, a restriction enzyme makes two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix.
After isolating the first restriction enzyme, HindII, in 1970[7], and the subsequent discovery and characterization of numerous restriction endonucleases,[8] the Nobel Prize in Medicine was awarded, in 1978, to Daniel Nathans, Werner Arber, and Hamilton Smith.[9] Their discovery led to the development of recombinant DNA technology that allowed, for example, the large scale production of human insulin for diabetics using E. coli bacteria.[10] Over 3000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially[11] and are routinely used for DNA modification and manipulation in laboratories
Restriction enzymes recognize a specific sequence of nucleotides[2] and produce a double-stranded cut in the DNA. While recognition sequences vary widely, with lengths between 4 and 8 nucleotides, many of them are palindromic, which correspond to nitrogenous base sequences that read the same backwards and forwards.[15] In theory, there are two types of palindromic sequences that can be possible in DNA. The mirror-like palindrome is similar to those found in ordinary text, in which a sequence reads the same forward and backwards on the same DNA strand (i.e., single stranded) as in GTAATG. The inverted repeat palindrome is also a sequence that reads the same forward and backwards, but the forward and backward sequences are found in complementary DNA strands (i.e., double stranded) as in GTATAC (Notice that GTATAC is complementary to CATATG)[16]. The inverted repeat is more common and has major biological importance than the mirror-like.
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EcoRI digestion produces “sticky” ends, whereas SmaI restriction enzyme cleavage produces “blunt” ends
Recognition sequences in DNA differ for each restriction enzyme, producing differences in the length, sequence and strand orientation (5′ end or the 3′ end) of a sticky-end “overhang” of an enzyme restriction.[17]
Different restriction enzymes that recognize the same sequence are known as neoschizomers. These often cleave in a different locales of the sequence; however, different enzymes which recognize and cleave in the same location are known as an isoschizomer.
Bacteria prevent their own DNA from being cut by modifying their nucleotides via DNA methylation
Restriction enzymes as tools
- See the main article on restriction digests.
Isolated restriction enzymes are used to manipulate DNA for different scientific applications.
They are used to assist insertion of genes into plasmid vectors during gene cloning and protein expression experiments. For optimal use, plasmids that are commonly used for gene cloning are modified to include a short polylinker sequence (called the multiple cloning site, or MCS) rich in restriction enzyme recognition sequences. This allows flexibility when inserting gene fragments into the plasmid vector; restriction sites contained naturally within genes influence the choice of endonuclease for digesting the DNA since it is necessary to avoid restriction of wanted DNA while intentionally cutting the ends of the DNA. To clone a gene fragment into a vector, both plasmid DNA and gene insert are typically cut with the same restriction enzymes, and then glued together with the assistance of an enzyme known as a DNA ligase.[27][28]
Restriction enzymes can also be used to distinguish gene alleles by specifically recognizing single base changes in DNA known as single nucleotide polymorphisms (SNPs).[29][30] This is only possible if a SNP alters the restriction site present in the allele. In this method, the restriction enzyme can be used to genotype a DNA sample without the need for expensive gene sequencing. The sample is first digested with the restriction enzyme to generate DNA fragments, and then the different sized fragments separated by gel electrophoresis. In general, alleles with correct restriction sites will generate two visible bands of DNA on the gel, and those with altered restriction sites will not be cut and will generate only a single band. The number of bands reveals the sample subject’s genotype, an example of restriction mapping.
In a similar manner, restriction enzymes are used to digest genomic DNA for gene analysis by Southern blot. This technique allows researchers to identify how many copies (or paralogues) of a gene are present in the genome of one individual, or how many gene mutations (polymorphisms) have occurred within a population. The latter example is called restriction fragment length polymorphism
