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EcoRI Restriction Enzyme Recognition Site
''Eco''RI (pronounced "eco R one") is a restriction endonuclease enzyme isolated from species ''E. coli.'' It is a restriction enzyme that cleaves DNA double helices into fragments at specific sites, and is also a part of the restriction modification system. The ''Eco'' part of the enzyme's name originates from the species from which it was isolated - "E" denotes generic name which is "Escherichia" and "co" denotes species name, "coli" - while the R represents the particular strain, in this case RY13, and the I denotes that it was the first enzyme isolated from this strain. In molecular biology it is used as a restriction enzyme. ''Eco''RI creates 4 nucleotide sticky ends with 5' end overhangs of AATT. The nucleic acid recognition sequence where the enzyme cuts is G↓AATTC, which has a palindromic, complementary sequence of CTTAA↓G. Other restriction enzymes, depending on their cut sites, can also leave 3' overhangs or blunt ends with no overhangs. Structure Primary struct ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, responding to stimuli, providing structure to cells and organisms, and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes, and which usually results in protein folding into a specific 3D structure that determines its activity. A linear chain of amino acid residues is called a polypeptide. A protein contains at least one long polypeptide. Short polypeptides, containing less than 20–30 residues, are rarely considered to be proteins and are commonly called peptides. The individual amino acid residues are bonded together by peptide bonds and adjacent amino acid residues. The sequence of amino acid residue ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Sticky And Blunt Ends
DNA ends refer to the properties of the ends of linear DNA molecules, which in molecular biology are described as "sticky" or "blunt" based on the shape of the complementary strands at the terminus. In sticky ends, one strand is longer than the other (typically by at least a few nucleotides), such that the longer strand has bases which are left unpaired. In blunt ends, both strands are of equal length – i.e. they end at the same base position, leaving no unpaired bases on either strand. The concept is used in molecular biology, in cloning, or when subcloning insert DNA into vector DNA. Such ends may be generated by restriction enzymes that break the molecule's phosphodiester backbone at specific locations, which themselves belong to a larger class of enzymes called exonucleases and endonucleases. A restriction enzyme that cuts the backbones of both strands at non-adjacent locations leaves a staggered cut, generating two overlapping sticky ends, while an enzyme that makes a str ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Restriction Enzymes
A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix. These enzymes are found in bacteria and archaea and provide a defense mechanism against invading viruses. Inside a prokaryote, the restriction enzymes selectively cut up ''foreign'' DNA in a process called ''restriction digestion''; meanwhile, host DNA is protected by a modification enzyme (a methyltransferase) that modifi ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Bacterial Enzymes
Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among the first life forms to appear on Earth, and are present in most of its habitats. Bacteria inhabit soil, water, acidic hot springs, radioactive waste, and the deep biosphere of Earth's crust. Bacteria are vital in many stages of the nutrient cycle by recycling nutrients such as the fixation of nitrogen from the atmosphere. The nutrient cycle includes the decomposition of dead bodies; bacteria are responsible for the putrefaction stage in this process. In the biological communities surrounding hydrothermal vents and cold seeps, extremophile bacteria provide the nutrients needed to sustain life by converting dissolved compounds, such as hydrogen sulphide and methane, to energy. Bacteria also live in symbiotic and parasitic relationships wi ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Molecular Biology
Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physical structure of biological macromolecules is known as molecular biology. Molecular biology was first described as an approach focused on the underpinnings of biological phenomena - uncovering the structures of biological molecules as well as their interactions, and how these interactions explain observations of classical biology. In 1945 the term molecular biology was used by physicist William Astbury. In 1953 Francis Crick, James Watson, Rosalind Franklin, and colleagues, working at Medical Research Council unit, Cavendish laboratory, Cambridge (now the MRC Laboratory of Molecular Biology), made a double helix model of DNA which changed the entire research scenario. They proposed the DNA structure based on previous research done by Ro ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
EcoRV
''Eco''RV (pronounced "eco R five") is a type II restriction endonuclease isolated from certain strains of ''Escherichia coli''. It has the alternative name Eco32I. In molecular biology, it is a commonly used restriction enzyme. It creates blunt ends. The enzyme recognizes the palindromic 6-base DNA sequence 5'-GAT, ATC-3' and makes a blunt end at the vertical line. The complementary sequence is then 3'-CTA, TAG-5'. The ends are blunt and can be ligated into a blunt cloning site easily but with lower efficiency than sticky ends. Structure The structure of this enzyme, and several mutants, in complex with the DNA sequence which it cuts has been solved by X-ray crystallography. The core of the enzyme consists of a five-stranded mixed β-sheet flanked by α-helices. The core is conserved in all other type II restriction endonucleases. It also has an N-terminal dimerization subdomain formed by a short α-helix, a two-stranded antiparallel -sheet, and a long α-helix. This subdom ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
EcoRII
Restriction endonuclease (REase) EcoRII (pronounced "eco R two") is an enzyme of restriction modification system (RM) naturally found in ''Escherichia coli'', a Gram-negative bacteria. Its molecular mass is 45.2 kDa, being composed of 402 amino acids. Mode of action EcoRII is a bacterial Type IIE REase that interacts with two othreeref name="pmid17845057"> copies of the pseudopalindromic DNA recognition sequence 5'- CCW GG- 3' (W = A or T), one being the actual target of cleavage, the other(s) serving as the allosteric activator(s). EcoRII cuts the target DNA sequence CCWGG, generating sticky ends. Cut diagram Structure The apo crystal structure of EcoRII mutant R88A () has been solved at 2.1 Å resolution. The EcoRII monomer has two domains, N-terminal and C-terminal, linked through a hinge loop. Effector-binding domain The N-terminal effector-binding domain has an archetypal DNA-binding pseudobarrel fold () with a prominent cleft. Structural superposition sho ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Star Activity
Star activity is the relaxation or alteration of the specificity of restriction enzyme mediated cleavage of DNA that can occur under reaction conditions that differ significantly from those optimal for the enzyme. The result is typically cleavage at non-canonical recognition sites, or sometimes complete loss of specificity. Differences which can lead to star include low ionic strength, high pH, and high (> 5% v/v) glycerol concentrations. The latter condition is of particular practical interest, since commercial restriction enzymes are usually supplied in a buffer containing a substantial amount of glycerol (50% v/v is typical), meaning insufficient dilution of the enzyme solution can cause star activity; this problem most often arises during double or multiple digests. Star activity can happen because of presence of Mg2+, as is seen in HindIII, for example. The term star activity was introduced by Mayer who characterized the modified activity in EcoRI. External links Star Acti ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Recombinant DNA
Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome. Recombinant DNA is the general name for a piece of DNA that has been created by combining at least two fragments from two different sources. Recombinant DNA is possible because DNA molecules from all organisms share the same chemical structure, and differ only in the nucleotide sequence within that identical overall structure. Recombinant DNA molecules are sometimes called chimeric DNA, because they can be made of material from two different species, like the mythical chimera. R-DNA technology uses palindromic sequences and leads to the production of sticky and blunt ends. The DNA sequences used in the construction of recombinant DNA molecules can originate from any species. For example, plant DNA may be joined to bacter ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
DNA Ligase
DNA ligase is a specific type of enzyme, a ligase, () that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. It plays a role in repairing single-strand breaks in duplex DNA in living organisms, but some forms (such as DNA ligase IV) may specifically repair double-strand breaks (i.e. a break in both complementary strands of DNA). Single-strand breaks are repaired by DNA ligase using the complementary strand of the double helix as a template, with DNA ligase creating the final phosphodiester bond to fully repair the DNA. DNA ligase is used in both DNA repair and DNA replication (see '' Mammalian ligases''). In addition, DNA ligase has extensive use in molecular biology laboratories for recombinant DNA experiments (see '' Research applications''). Purified DNA ligase is used in gene cloning to join DNA molecules together to form recombinant DNA. Enzymatic mechanism The mechanism of DNA ligase is to form two covalent phos ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Clone (genetics)
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word ''cloning'' refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine. In a conventional molecular cloning experiment, the DNA to be cloned is obtained from an organism of interest, then treated with enzymes in the test tube to generate smaller DNA fragments. Subsequently, these fragments are then combined with vector DNA to generate recombinant DNA molecules. The recombinant DNA is then in ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
