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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 ''Eco''RI (pronounced "e ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Restriction Enzyme
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 mod ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Ionic Strength
The ionic strength of a solution is a measure of the concentration of ions in that solution. Ionic compounds, when dissolved in water, dissociate into ions. The total electrolyte concentration in solution will affect important properties such as the dissociation constant or the solubility of different salts. One of the main characteristics of a solution with dissolved ions is the ionic strength. Ionic strength can be molar (mol/L solution) or molal (mol/kg solvent) and to avoid confusion the units should be stated explicitly. The concept of ionic strength was first introduced by Lewis and Randall in 1921 while describing the activity coefficients of strong electrolytes. Quantifying ionic strength The molar ionic strength, ''I'', of a solution is a function of the concentration of ''all'' ions present in that solution. :I = \begin\frac\end\sum_^ c_i z_i^ where one half is because we are including both cations and anions, ''c''i is the molar concentration of ion i (M, mol ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Glycerol
Glycerol (), also called glycerine in British English and glycerin in American English, is a simple triol compound. It is a colorless, odorless, viscous liquid that is sweet-tasting and non-toxic. The glycerol backbone is found in lipids known as glycerides. Because it has antimicrobial and antiviral properties, it is widely used in wound and burn treatments approved by the U.S. Food and Drug Administration. Conversely, it is also used as a bacterial culture medium. It can be used as an effective marker to measure liver disease. It is also widely used as a sweetener in the food industry and as a humectant in pharmaceutical formulations. Because of its three hydroxyl groups, glycerol is miscible with water and is hygroscopic in nature. Structure Although achiral, glycerol is prochiral with respect to reactions of one of the two primary alcohols. Thus, in substituted derivatives, the stereospecific numbering labels the molecule with a "sn-" prefix before the stem name of ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Buffer Solution
A buffer solution (more precisely, pH buffer or hydrogen ion buffer) is an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa. Its pH changes very little when a small amount of strong acid or base is added to it. Buffer solutions are used as a means of keeping pH at a nearly constant value in a wide variety of chemical applications. In nature, there are many living systems that use buffering for pH regulation. For example, the bicarbonate buffering system is used to regulate the pH of blood, and bicarbonate also acts as a buffer in the ocean. Principles of buffering Buffer solutions resist pH change because of a chemical equilibrium between the weak acid HA and its conjugate base A−: When some strong acid is added to an equilibrium mixture of the weak acid and its conjugate base, hydrogen ions (H+) are added, and the equilibrium is shifted to the left, in accordance with Le Chatelier's principle. Because of this, the hydrogen io ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
HindIII
''Hin''dIII (pronounced "Hin D Three") is a type II site-specific deoxyribonuclease restriction enzyme isolated from ''Haemophilus influenzae'' that cleaves the DNA palindromic sequence AAGCTT in the presence of the cofactor Mg2+ via hydrolysis. The cleavage of this sequence between the AA's results in 5' overhangs on the DNA called sticky ends: 5'-A , A G C T T-3' 3'-T T C G A, A-5' Restriction endonucleases are used as defense mechanisms in prokaryotic organisms in the restriction modification system. Their primary function is to protect the host genome against invasion by foreign DNA, primarily bacteriophage DNA. There is also evidence that suggests the restriction enzymes may act alongside modification enzymes as selfish elements, or may be involved in genetic recombination and transposition. Enzyme Structure The structure of HindIII is complex, and consists of a homodimer. Like other type II restriction endonucleases, it is believed to contain ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
EcoRI
''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 s ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
New England Biolabs
New England Biolabs (NEB) produces and supplies recombinant and native enzyme reagents for the life science research, as well as providing products and services supporting genome editing, synthetic biology and next-generation sequencing. NEB also provides free access to research tools such as REBASE, InBASE, and Polbase. The company The company was established in 1974 by Donald Comb as a cooperative laboratory of experienced scientists and initially produced restriction enzymes on a commercial scale. The company then began producing solution-oriented products. It received approximately $1.7 million in Small Business Innovation Research (SBIR) grants between 2009 and 2013 for this research. NEB produces 230 recombinant and 30 native restriction enzymes for genomic research, as well as nicking enzymes and DNA methylases. It pursues research in areas related to proteomics, DNA Sequencing, and drug discovery. NEB scientists also conduct basic research in Molecular Biolog ... [...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 mod ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
