Restriction enzyme digestion principle
To analyze genomic DNA, we must first cut it into smaller, more manageable pieces. The tools for this are restriction enzymes. A restriction enzyme (or restriction endonuclease) recognizes a specific nucleotide-pair sequence in DNA called a restriction site and cleaves the DNA (hydrolyzes the phosphodiester backbones) within or near that sequence. All restriction enzymes cut DNA between the carbon and the phosphate moiety of the phosphodiester bond so that fragments produced by restriction enzyme digestion have phosphates and hydroxyls. Most restriction enzymes function optimally at Restriction enzymes are used to produce a pool of DNA fragments to be cloned. Restriction enzymes are also used to analyze the positions of restriction sites in a piece of cloned DNA or in a segment of DNA in the genome. In most laboratory uses of restriction enzyme digestions (usually shortened to restriction digests), we attempt to “cut to completion,” meaning that the enzyme is allowed to cut at every one of its restriction sites in the DNA. Such a digest will cut each genome copy of the same organism into the same large set of pieces. As we will see, in certain genomics applications it is desirable, instead, to do a “partial digest” in which the 37°C.
Enzyme does not have enough time to complete its job. As a result, only some of the restriction sites are cut, and many are left uncut. Because we are cutting millions of identical DNA molecules, in a partial digest each will be cut at a unique subset of the available restriction sites.
Most restriction enzymes are found naturally in bacteria, although a handful have been found in eukaryotes. In bacteria, restriction enzymes protect the host organism against viruses by cutting up—restricting—invading viral DNA. The bacterium modifies its own restriction sites (by methylation) so that its own DNA is protected from the action of the restriction enzyme(s) it makes. Werner Arber, Daniel Nathans, and Hamilton O. Smith received the 1978 Nobel Prize in Physiology or Medicine “for their discovery of restriction enzymes and their application in problems of molecular genetics.” More than 400 different restriction enzymes have been isolated, and at least 2,000 more have been characterized partially. They are named for the organisms from which they are isolated. Conventionally, a three-letter system is used. Commonly the first letter is that of the genus, and the second and third letters are from the species name. The letters are italicized or underlined, followed by roman numerals that signify a specific restriction enzyme from that organism. Additional letters sometimes are added just before the number to signify a particular bacterial strain from which the enzymes were obtained. For example, EcoRI and EcoRV are both from Escherichia coli strain RY13, but recognize different restriction sites; HindIII is from Haemophilus influenzae strain Rd. The Roman numerals indicate the order in which the restriction enzymes from that strain were identified. Hence, EcoRI and EcoRV are the first and fifth restriction enzymes identified for E. coli strain RY13. The names are pronounced in ways that follow no set pattern. For example, BamHI is “bam-H-one,” BglII is “bagel-two,” EcoRI is “Echo-R-one” or “eeko-R-one,” HindIII is “hin-D-three,” HhaI is “ha-ha-one,” and HpaII is “hepa-two.”
Many restriction sites have an axis of symmetry through the midpoint. Figure 8.1 shows this symmetry for the EcoRI restriction site: the nucleotide sequence from to on one DNA strand is the same as the nucleotide sequence from to on the complementary DNA strand. Thus, the sequences are said to have twofold rotational symmetry. A number of restriction sites are shown in Table 8.1. The most commonly used restriction enzymes recognize four nucleotide pairs (for example, HhaI) or six nucleotide pairs (for example, BamHI, EcoRI). Some enzymes recognize eight-nucleotide pair sequences (for example, NotI [“not-one”]). Other classes of enzymes do not fit our model because the restriction site is not symmetrical about the center. HinfI (“hin-fone”), for example, recognizes a five-nucleotide pair sequence in which there is symmetry in the two nucleotide pairs on either side of the central nucleotide pair, but the central nucleotide pair is obviously asymmetrical within the sequence. BstXI (“b-s-t-x-one”) is representative of a number of restriction enzymes with a nonspecific spacer region between symmetrical sequences.
Enzyme does not have enough time to complete its job. As a result, only some of the restriction sites are cut, and many are left uncut. Because we are cutting millions of identical DNA molecules, in a partial digest each will be cut at a unique subset of the available restriction sites.
Most restriction enzymes are found naturally in bacteria, although a handful have been found in eukaryotes. In bacteria, restriction enzymes protect the host organism against viruses by cutting up—restricting—invading viral DNA. The bacterium modifies its own restriction sites (by methylation) so that its own DNA is protected from the action of the restriction enzyme(s) it makes. Werner Arber, Daniel Nathans, and Hamilton O. Smith received the 1978 Nobel Prize in Physiology or Medicine “for their discovery of restriction enzymes and their application in problems of molecular genetics.” More than 400 different restriction enzymes have been isolated, and at least 2,000 more have been characterized partially. They are named for the organisms from which they are isolated. Conventionally, a three-letter system is used. Commonly the first letter is that of the genus, and the second and third letters are from the species name. The letters are italicized or underlined, followed by roman numerals that signify a specific restriction enzyme from that organism. Additional letters sometimes are added just before the number to signify a particular bacterial strain from which the enzymes were obtained. For example, EcoRI and EcoRV are both from Escherichia coli strain RY13, but recognize different restriction sites; HindIII is from Haemophilus influenzae strain Rd. The Roman numerals indicate the order in which the restriction enzymes from that strain were identified. Hence, EcoRI and EcoRV are the first and fifth restriction enzymes identified for E. coli strain RY13. The names are pronounced in ways that follow no set pattern. For example, BamHI is “bam-H-one,” BglII is “bagel-two,” EcoRI is “Echo-R-one” or “eeko-R-one,” HindIII is “hin-D-three,” HhaI is “ha-ha-one,” and HpaII is “hepa-two.”
Many restriction sites have an axis of symmetry through the midpoint. Figure 8.1 shows this symmetry for the EcoRI restriction site: the nucleotide sequence from to on one DNA strand is the same as the nucleotide sequence from to on the complementary DNA strand. Thus, the sequences are said to have twofold rotational symmetry. A number of restriction sites are shown in Table 8.1. The most commonly used restriction enzymes recognize four nucleotide pairs (for example, HhaI) or six nucleotide pairs (for example, BamHI, EcoRI). Some enzymes recognize eight-nucleotide pair sequences (for example, NotI [“not-one”]). Other classes of enzymes do not fit our model because the restriction site is not symmetrical about the center. HinfI (“hin-fone”), for example, recognizes a five-nucleotide pair sequence in which there is symmetry in the two nucleotide pairs on either side of the central nucleotide pair, but the central nucleotide pair is obviously asymmetrical within the sequence. BstXI (“b-s-t-x-one”) is representative of a number of restriction enzymes with a nonspecific spacer region between symmetrical sequences.
Frequency of Occurrence of Restriction Sites in DNA. Since each restriction enzyme cuts DNA at an enzyme-specific sequence, the number of cuts the enzyme makes in a particular DNA molecule depends on the number of times that particular restriction site occurs. When we cut a number of copies of the same genome with a particular restriction enzyme, the DNA is cleaved at the specific restriction sites for the enzyme, which are distributed throughout the genome. Although this produces millions of fragments of different sizes from one genome copy, all copies of the same genome will be cut at identical places Based on probability principles, the frequency of a short nucleotide pair sequence in the genome theoretically will be greater than the frequency of a long nucleotide pair sequence, so an enzyme that recognizes a four-nucleotide pair sequence will cut a DNA molecule more frequently than one that recognizes a six-nucleotide pair sequence, and both enzymes will cut more frequently than one that recognizes an eight-nucleotide pair sequence.
Summary:
Summary:
- Restriction enzyme acts as Endonuclease on foreign ds DNA. This enzyme recognize recognition sequence and bind at that position. Recognition site can 4 nucleotide sequences, 6 nucleotide sequences or (8 nucleotide sequences it is very rare).
- Restriction enzyme cleave foreign ds DNA segment at specific site of nucleotide sequence, generally this sequence is GAATTC. It cleave between A-T nucleotide for cleavage.
- Bacterial chromosomes have segment of ds DNA is going to produce Restriction enzyme i.e.RE1 and RE2 whenever bacteriophage is going to infect.
- It have activity of restriction medication ability because when ever bacterial cell is going to fight against foreign invader i.e. bacteriophage. There are chance to cleave their own DNA segment, so that Bacterial own DNA get Methylated to prevent cleavage of their own DNA and that’s why restriction cleave foreign DNA and chop it to destroy.
- Either cleavage or Modification both the thinks are accomplished by restriction enzyme.
- There are four types of restriction enzyme Such as Type-I, Type-II, Type-III, Type-IIs
- Type-I cleave 1000base pair upstream position from recognition site, it having ability to distant cutter and multisubunit single enzyme.
- Type-II it is multienzyme complex it having the activity of cleavage and modification. Cleavage site is the recognition site on DNA.
- Type-III it cleaves 26 base pair upstream position from recognition site. multisubunit single enzyme
- Type-IIs Cleavage site is the recognition site on DNA but it having ability to distant cutter and multisubunit single enzyme.
- Recognition sequence is same but the pattern of cleavage is different is known as Neoschizomers
- That restriction enzyme isolated from different source but it having same cleavage site that restriction enzyme is called Isoschizomer.
- There are some restriction enzyme can generate sticky or blunt end.