Restriction Enzyme

Restriction enzymes (RE) are endonucleases that recognize specific Deoxyribonucleic acid sequences between iv and viii bp long and typically carve the strands at a specific and constant position inside or earlier the recognition site.

From: Stock Identification Methods (Second Edition) , 2014

Recombinant DNA Engineering

David P. Clark , Nanette J. Pazdernik , in Biotechnology (Second Edition), 2016

Restriction enzymes are useful for many unlike applications. Because the DNA sequence is different in each organism, the design of restriction sites volition also be different. The source of isolated Deoxyribonucleic acid can be identified by this blueprint. If genomic Deoxyribonucleic acid is isolated from 1 organism and cut with i particular brake enzyme, a specific set of fragments tin can be separated and identified past electrophoresis. If DNA from a different organism is cut by the aforementioned restriction enzyme, a different ready of fragments will be generated. This technique tin be applied to Dna from two individuals from the aforementioned species. Although the DNA sequence differences will be small, restriction enzymes tin can exist used to place these differences. If the sequence divergence falls in a restriction enzyme recognition site, it gives a restriction fragment length polymorphism (RFLP) (Fig. A). When the restriction enzyme patterns are compared, the number and size of one or 2 fragments will be afflicted for each base of operations divergence that affects a cut site.

FIGURE A. RFLP Assay

Dna from related organisms shows small differences in sequence that cause changes in brake sites. In the example shown, cutting a segment of Deoxyribonucleic acid from the first organism yields six fragments of different sizes (labeled a–f on the gel). If the equivalent region of DNA from a related organism were digested with the same enzyme, a similar blueprint would exist expected. Here, a single-nucleotide difference is present, which eliminates ane of the restriction sites. Consequently, digesting this Dna produces only five fragments. Fragments c and d are no longer seen but form a new ring labeled cd.

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Transgenic Plants

Francisco Murilo Zerbini , ... Marcos Fernando Basso , in Biotechnology and Institute Breeding, 2014

Restriction Enzymes

Restriction enzymes, also called brake endonucleases, recognize a specific sequence of nucleotides in double stranded Dna and cutting the Dna at a specific location. They are indispensable to the isolation of genes and the construction of cloned DNA molecules. Most brake enzymes recognize sequences of 4 to eight base pairs and hydrolyze a single phosphodiester bond on each strand. A feature of many of these cleavage or restriction sites is their double rotational symmetry. Generally, the cleavage sites are symmetrically positioned, or palindromic. Restriction enzymes tin can create fragments with sticky ends, equally is the instance with the enzyme BamHI, or blunt ends, as with Hae3 (Table 8.i).

Table eight.one. The Specificity of Some Restriction Enzymes

Name Origin Recognition Sequence
BamHowdy Bacillus amyloliquefaciens H v′ Grand//GATCC iii′
three′ CCTAG//M five′
EcoRI Escherichia coli RY13 five′ Thousand//AATTC three′
three″CTTAA//G 5′
HindIII Haemophilus influenzae Rd 5′ A//AGCTT 3′
3′ TTCGA//A 5′
HaeIII Haemophilus aegyptius 5′ GG//CC 3′
3′ CC//GG 5′
KpnI Klebsiella pneumoniae v′ GGTAC//C three′
3′ C//CATGG 5′

Double bars indicate the cleavage site in the Deoxyribonucleic acid strand.

DNA ligases are used to join the fragments of Deoxyribonucleic acid generated by restriction enzymes. The availability of various types of brake enzymes and ligases enables the transfer of specific DNA sequences from one molecule to another.

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The Cell

Tetsuya Watanabe , in Biophysical Basis of Physiology and Calcium Signaling Mechanism in Cardiac and Smooth Muscle, 2018

4.xviii Restriction Enzymes

Restriction enzymes of leaner catalyze the cleavage of a foreign Deoxyribonucleic acid such equally those injected by a phage (a virus that infects leaner). Leaner acquired those enzymes in society to defend themselves against such invasions. Each restriction enzyme cuts DNA at a specific recognition sequence. For example, the enzyme EcoRI and DraI cut Deoxyribonucleic acid only where the specific paired sequence is encountered equally shown in Fig. four.25. Today, some 276 restriction enzymes have been known.

Fig. 4.25

Fig. iv.25. Restriction enzymes cutting DNA bonds betwixt iii′ OH of one nucleotide and 5′ phosphate of the next ane at the specific restriction site.

Adding methyl groups to certain bases at the recognition sites on the bacterial DNA blocks the restriction enzyme to bind and protects the bacterial Deoxyribonucleic acid from being cut by themselves. Those methylases in the host might also modify a few phage DNA, which in turn help the phage escape the restriction. The few phages that accept escaped from one strain of leaner acquired the resistance for the strain of bacteria and can and so grow well on a new host [12]. DNA molecules of unlike species cut by the same restriction enzyme can be pasted together by the enzyme called a ligase.

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Deoxyribonucleic acid and Aspects of Molecular Biology

Kent S. Gates , in Comprehensive Natural Products Chemical science, 1999

7.xiv.ii.19 Restriction and Methylation Enzymes

Brake enzymes catalyze the sequence-specific hydrolysis of double-stranded Deoxyribonucleic acid. Restriction enzymes occur in microorganisms as part of brake–methylation systems consisting of Dna-cleaving enzyme/DNA-methylating enzyme pairs that recognize a common sequence. Sequence-specific methylation of its own DNA protects the host against DNA degradation by its own restriction enzymes. Brake–modification systems appear to serve as a prokaryotic immune system that protects these organisms against foreign Deoxyribonucleic acid that might enter the cell. 379 The enzymes of restriction–methylation systems in microorganisms formally can exist considered secondary metabolite natural products. The chemistry and enzymology of brake–methylation enzyme systems has been reviewed. 379,380

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Neurobiology of Steroids

Michael Karl , ... George P. Chrousos , in Methods in Neurosciences, 1994

Restriction Enzyme Analysis

Brake enzymes are enzymes that demark to specific recognition sequences to cleave double-stranded DNA (38). Mutations creating or abolishing such recognition sites tin can, therefore, be investigated by employment of brake enzymes. In one case a change in a recognition site has been identified in the DNA sequence of a patient, specific PCR-amplified fragments of the Deoxyribonucleic acid obtained from relatives or controls, potentially harboring the base of operations change, are incubated with the specific restriction enzyme. The cut or uncut segments can be visualized by gel electrophoresis to decide whether an individual is a carrier of such a mutation or bears the wild-type sequence. Unfortunately, only a small number of mutations can be identified past this technique.

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Brake Endonucleases and Modification Methylases

Hyone-Myong Eun , in Enzymology Primer for Recombinant Dna Technology, 1996

iv. Deoxyribonucleic acid Conformation.

Restriction enzymes are not simply sequence specific simply also structure sensitive and may exhibit either enhanced or inhibited cleavage activity. The restriction sites located in nuclease SI-sensitive, conformationally flexible junctions, for example, between contiguous B-DNA and Z-Deoxyribonucleic acid or in non-B-DNA conformation, are resistant to cleavage by many restriction enzymes such as BamHello, EcoRI, HindIII, and PstI (6, vii). The 5′-GCGC-3′ site of HhaI is not cleaved when the sequence is inside the stretch of alternating GC sequences that adopt a Z-DNA conformation (viii). In contrast, MboI (GATC) has an enhanced cleavage reactivity at the B-Z junction (ix). Modulation of Deoxyribonucleic acid conformation by Deoxyribonucleic acid intercalating drugs, oligonucleotides, or oligonucleotide analogs thus provides an efficient mode for achieving controlled cleavages by brake enzymes (see below).

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DNA Methylome of Endometrial Cancer

Golnaz Asaadi Tehrani , in Computational Epigenetics and Diseases, 2019

Enzyme Digestion-Based Methods

Restriction enzyme : Methylation-sensitive restriction enzymes (MREs) such as BstU1, Hpa Ii, Not1, and SmaI cleavage simply unmethylated target sequences, and methylated Dna remains intact; Dna fragments are size selected, then sequencing technologies predict genome-broad DNA methylation level.

Comparative loftier-throughput arrays of relative methylation (Charm) uses McrBC enzyme, in which it recognizes RmC(Due north)55–103RmC sites, which cleaves half of the methylated DNAs and all the methylated CGIs methylated Deoxyribonucleic acid, later on uses array hybridization. This method is able to detect differentially methylated regions (DMRs) at CGI shores, which are otherwise not detectable with CpG-directed enrichment methods, such equally methylated Dna immunoprecipitation (MeDIP).

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Approaches to Detecting Dna Base Modification in the Encephalon

X. Li , Westward. Wei , in Deoxyribonucleic acid Modifications in the Encephalon, 2017

Restriction Enzymes for DNA Modifications

Restriction enzymes are ane of the easiest approaches to detect modified Dna at specific genomic sites. Cleavage of DNA past a restriction enzyme may be blocked or impaired when a particular base in the recognition site is modified. For case, MspI and HpaII recognize the aforementioned sequence (CCGG); withal, they are sensitive to different modification status: when the external C in the sequence CCGG is methylated, MspI and HpaII cannot carve. Different HpaII, MspI can cleave the sequence when the internal C residue is methylated (Bird & Southern, 1978). Another enzyme, Pvurts1I, only cleave the sequence hmCN11–12/N9–10M, which contains 5hmC (Asgar Abbas, Monika, Honorata, & Matthias, 2014; Evelina & Giedrius, 2014; Sun et al., 2015). The combination of DpnI and DpnII is use to discover m6dA; both recognize the consensus sequence GATC, but only DpnI will cleave at this site if the adenine is methylated (Fu et al., 2015; Greer et al., 2015; Heyn & Esteller, 2015; Ratel, Ravanat, Berger, & Wion, 2006). Thus, using different restriction enzymes, we could discover DNA modification beyond familiar cytosine, including 5mC and 5hmC. In addition, this approach is cost-effective and fast; notwithstanding, information technology is still limited by the number and distribution of brake sites in the genome. Dai et al. (2002) plant that a maximum of 4100 sites can exist accessed by the restriction enzymes known to be Deoxyribonucleic acid modification sensitive, and specific sites of involvement that are non located at restriction sites cannot be investigated using this method.

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Molecular Systematics and the Evolution of Arthropods

Marjorie A. Hoy , in Insect Molecular Genetics (Third Edition), 2013

12.4.3 Restriction Fragment Length Polymorphism (RFLP) Analysis

Restriction-enzyme analyses are versatile, providing information on the nature, as well as the extent, of differences between sequences in nuclear or mitochondrial DNA (mtDNA) (Dowling et al. 1996, Table 12.3 ). RFLP analyses reveal variations inside a species in the length of Deoxyribonucleic acid fragments generated by a specific restriction endonuclease. RFLP variations are caused by mutations that create or eliminate recognition sites for the restriction enzymes.

RFLP analyses tin can be used finer, and relatively economically, to clarify clonal populations, heterozygosity, relatedness, geographic variation, hybridization, species boundaries, and phylogenies ranging in age from 0 to l   mya (Table 12.3). It is possible to analyze more loci per individual by RFLP analysis than by Deoxyribonucleic acid sequencing considering RFLPs are less fourth dimension-consuming and expensive, although the data provided for each locus is less consummate (Dowling et al. 1996, Hall 1998). Higher level systematics studies only rarely have used RFLPs.

More than 1400 restriction enzymes are known that cut Deoxyribonucleic acid at a specific position inside a specific recognition sequence. See Chapter 5 for a word of restriction digests, also every bit Chocolate-brown (1991), and catalogs from a variety of commercial producers. Most recognition sequences are 4–six   bp long, although they can be every bit large as 12   bp. The specificity of restriction enzymes ways that a complete digestion volition yield a reproducible array of DNA fragments. Changes in the number and size of fragments can occur past changes in DNA sequence past rearrangements (inversions, tandem duplication, and inverted duplication), or addition, deletion, or exchange of specific bases.

Once the DNA is digested with a restriction enzyme, the fragments produced are sorted by size using agarose or polyacrylamide gel electrophoresis. Dna fragments of known length are run on each gel to serve as an internal standard and to allow the size of the experimental fragments to be estimated. The Dna fragments in the gel are visualized by several methods, including staining with ethidium bromide (if the DNA was previously amplified by the PCR), or by probing Southern blots with labeled probes. The detection technique used depends on the amount of Dna present in the gel.

Staining with ethidium bromide is simple and cheap, but least sensitive. The minimal corporeality of Dna in a band that can be detected by ethidium bromide is ≈2   ng, then small fragments can be detected only if a large amount of DNA is present. DNA probes can be end labeled by adding 32P-labeled nucleotides to the ends of Deoxyribonucleic acid fragments produced by the restriction enzymes. Intensity of labeling is independent of fragment size and is more than sensitive than ethidium bromide (EtBr), with 1–v   ng of Deoxyribonucleic acid easily visualized. If primers are available, DNA can exist outset amplified by the PCR, cut with a restriction enzyme, and labeled by ethidium bromide (PCR-RFLP).

If less DNA is bachelor, radiolabeled DNA probes can be used to visualize fragments. Southern-absorb hybridizations are highly sensitive and picogram quantities of Dna can be detected, although small fragments <fifty   bp in size are more difficult to detect. Southern blots require a suitable probe with sufficient sequence similarity to the target DNA that a stable hybrid can be formed at moderate to loftier stringency. The use of probes from other species (heterologous probes) makes interpretation of results more difficult.

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FGF Mutants

Xiaokun Li , ... Zhiyong Sheng , in Fibroblast Growth Factors, 2018

two.1 Reagent

Restriction enzymes NdeI, BamHI, and BglIi were purchased from NEB Company (Us); Pyrobest Deoxyribonucleic acid polymerase was from Dalian Takara Company (Prc); PCR puriWcation kit, gel extraction kit, and plasmid miniprep kit were obtained from Shanghai Biocolors Company (Communist china); CM–Sepharose and heparin–Sepharose were from Pharmacia (The states); The plasmid pUC-haFGF, expression vector pET3c, and Escherichia coli strain BL21(DE3) were kept by Biopharmaceutical Enquiry and Development Center of Jinan University; Primers were synthesized by Shanghai Sangon Company (China).

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