Thermodynamics | Turkish Chemistry
May 11

Restriction enzyme

A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded or single stranded at specific recognition nucleotide sequences known as restriction sites.[1][2][3] Such enzymes, found in 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 in a process called restriction; host 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 , a restriction enzyme makes two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the 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 technology that allowed, for example, the large scale production of human insulin for diabetics using E. coli .[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 modification and manipulation in laboratories

Restriction enzymes recognize a specific sequence of nucleotides[2] and produce a double-stranded cut in the . While recognition sequences vary widely, with lengths between 4 and 8 nucleotides, many of them are palindromic, which correspond to nitrogenous sequences that read the same backwards and forwards.[15] In theory, there are two types of palindromic sequences that can be possible in . 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 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 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.

 

EcoRI digestion produces “sticky” ends, whereas SmaI restriction enzyme cleavage produces “blunt” ends

Recognition sequences in 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.

prevent their own from being cut by modifying their nucleotides via methylation

Restriction enzymes as tools

See the main article on restriction digests.

Isolated restriction enzymes are used to manipulate for different scientific applications.

They are used to assist insertion of genes into plasmid vectors during gene cloning and 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 since it is necessary to avoid restriction of wanted while intentionally cutting the ends of the . To clone a gene fragment into a vector, both plasmid and gene insert are typically cut with the same restriction enzymes, and then glued together with the assistance of an enzyme known as a ligase.[27][28]

Restriction enzymes can also be used to distinguish gene alleles by specifically recognizing single changes in 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 sample without the need for expensive gene sequencing. The sample is first digested with the restriction enzyme to generate fragments, and then the different sized fragments separated by gel electrophoresis. In general, alleles with correct restriction sites will generate two visible bands of 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 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

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Chemical kinetics
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Chemical kinetics, also known as reaction kinetics, is the study of rates of chemical processes. Chemical kinetics includes investigations of how different experimental conditions can influence the speed of a chemical reaction and yield information about the reaction’s mechanism and transition states, as well as the construction of mathematical models that can describe the characteristics of a chemical reaction. In 1864, Peter Waage and Cato Guldberg pioneered the development of chemical kinetics by formulating the law of mass action, which states that the speed of a chemical reaction is proportional to the quantity of the reacting substances.

Chemical kinetics deals with the experimental determination of reaction rates from which rate laws and rate constants are derived. Relatively simple rate laws exist for zero order reactions (for which reaction rates are independent of concentration), first order reactions, and second order reactions, and can be derived for others. In consecutive reactions the rate-determining step often determines the kinetics. In consecutive first order reactions, a steady state approximation can simplify the rate law. The activation for a reaction is experimentally determined through the Arrhenius equation and the Eyring equation. The main factors that influence the reaction rate include: the physical state of the , the concentrations of the , the temperature at which the reaction occurs, and whether or not any catalysts are present in the reaction.

Nature of the

Depending upon what substances are reacting, the time varies. Acid reactions, the formation of salts, and ion exchange are fast reactions. When covalent bond formation takes place between the molecules and when large molecules are formed, the reactions tend to be very slow.

Physical state

The physical state (solid, liquid, or gas) of a reactant is also an important factor of the rate of change. When are in the same phase, as in aqueous solution, thermal motion brings them into contact. However, when they are in different phases, the reaction is limited to the interface between the . Reaction can only occur at their area of contact, in the case of a liquid and a gas, at the surface of the liquid. Vigorous shaking and stirring may be needed to bring the reaction to completion. This means that the more finely divided a solid or liquid reactant, the greater its surface area per unit volume, and the more contact it makes with the other reactant, thus the faster the reaction. To make an analogy, for example, when one starts a fire, one uses wood chips and small branches—one doesn’t start with large logs right away. In organic On reactions are the exception to the rule that homogeneous reactions take place faster than heterogeneous reactions

Concentration

Concentration plays a very important role in reactions according to the collision theory of chemical reactions, because molecules must collide in order to react together. As the concentration of the increases, the frequency of the molecules colliding increases, striking each other more frequently by being in closer contact at any given point in time. Think of two being in a closed container. All the molecules contained within are colliding constantly. By increasing the amount of one or more of the it causes these collisions to happen more often, increasing the reaction rate

Temperature

Temperature usually has a major effect on the rate of a chemical reaction. Molecules at a higher temperature have more thermal . Although collision frequency is greater at higher temperatures, this alone contributes only a very small proportion to the increase in rate of reaction. Much more important is the fact that the proportion of reactant molecules with sufficient to react ( greater than activation : E > Ea) is significantly higher and is explained in detail by the Maxwell–Boltzmann distribution of molecular energies.

The ‘rule of thumb’ that the rate of chemical reactions doubles for every 10° C temperature rise is a common misconception. This may have been generalized from the special case of biological systems, where the Q10 (temperature coefficient) is often between 1.5 and 2.5.

A reaction’s kinetics can also be studied with a temperature jump approach. This involves using a sharp rise in temperature and observing the relaxation rate of an equilibrium process.

Catalysts

A catalyst is a substance that accelerates the rate of a chemical reaction but remains chemically unchanged afterwards. The catalyst increases rate reaction by providing a different reaction mechanism to occur with a lower activation . In autocatalysis a reaction product is itself a catalyst for that reaction leading to positive feedback. Proteins that act as catalysts in biochemical reactions are called enzymes. Michaelis-Menten kinetics describe the rate of enzyme mediated reactions.A catalyst does not affect the position of the equilibria, as the catalyst speeds up the backward and forward reactions equally.

In certain organic molecules specific substituents can have an influence on reaction rate in neighbouring group participation.

Agitating or mixing a solution will also accelerate the rate of a chemical reaction, as this gives the particles greater , increasing the number of collisions between and therefore the possibility of successful collisions.

Pressure

Increasing the pressure in a gaseous reaction will increase the number of collisions between , increasing the rate of reaction. This is because the activity of a gas is directly proportional to the partial pressure of the gas. This is similar to the effect of increasing the concentration of a solution

Equilibrium

While chemical kinetics is concerned with the rate of a chemical reaction, thermodynamics determines the extent to which reactions occur. In a reversible reaction, chemical equilibrium is reached when the rates of the forward and reverse reactions are equal and the concentrations of the and products no longer change. This is demonstrated by, for example, the Haber–Bosch process for combining nitrogen and hydrogen to produce ammonia. Chemical clock reactions such as the Belousov–Zhabotinsky reaction demonstrate that component concentrations can oscillate for a long time before finally attaining the equilibrium

Free

In general terms, the free change (ΔG) of a reaction determines if a chemical change will take place, but kinetics describes how fast the reaction is. A reaction can be very exothermic and have a very positive entropy change but will not happen in practice if the reaction is too slow. If a reactant can produce two different products, the thermodynamically most stable one will generally form except in special circumstances when the reaction is said to be under reaction control. The Curtin–Hammett principle applies when determining the product ratio for two interconverting rapidly, each going to a different product. It is possible to make predictions about reaction rate constants for a reaction from free- relationships.

The isotope effect is the difference in the rate of a chemical reaction when an atom in one of the is replaced by one of its isotopes.

Chemical kinetics provides information on residence time and heat transfer in a chemical reactor in chemical engineering and the molar mass distribution in polymer .

Applications

The mathematical models that describe chemical reaction kinetics provide chemists and chemical engineers with tools to better understand and describe chemical processes such as food decomposition, microorganism growth, stratospheric ozone decomposition, and the complex of biological systems. These models can also be used in the design or modification of chemical reactors to optimize product yield, more efficiently separate products, and eliminate environmentally harmful by-products. When performing catalytic cracking of heavy hydrocarbons into gasoline and light gas, for example, models can be used to find the temperature and pressure at which the highest yield of heavy hydrocarbons into gasoline will occur.

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