· DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR.Transcriptome analysis reveals that constant heat stress modifies the metabolism and structure of the porcine longissimus dorsi skeletal muscle. Q.1-The monomeric deoxyribonucleotide units of DNA include all except-A) Deoxyadenylate, B) Deoxyguanylate. C) Deoxycytidylate, D) Deoxyuridylate. DNA polymerase - Wikipedia. In molecular biology, DNA polymerases are enzymes that synthesize DNAmolecules from deoxyribonucleotides, the building blocks of DNA. These enzymes are essential to DNA replication and usually work in pairs to create two identical DNA strands from a single original DNA molecule. During this process, DNA polymerase "reads" the existing DNA strands to create two new strands that match the existing ones.[1][2][3][4][5][6]These enzymes catalyze the following chemical reactiondeoxynucleoside triphosphate + DNAn ⇌ diphosphate + DNAn+1. Catalyses DNA- template- directed extension of the 3'- end of a DNA strand by one nucleotide at a time. Every time a cell divides, DNA polymerases are required to help duplicate the cell's DNA, so that a copy of the original DNA molecule can be passed to each daughter cell. In this way, genetic information is passed down from generation to generation. Before replication can take place, an enzyme called helicase unwinds the DNA molecule from its tightly woven form, in the process breaking the hydrogen bonds between the annealed nucleotide bases. This opens up or "unzips" the double- stranded DNA to give two single strands of DNA that can be used as templates for replication. Contents. 1History. Function. 3Variation across species. Prokaryotic DNA polymerases. Eukaryotic DNA polymerase. Polymerases β, λ, σ and μ (beta, lambda, sigma, and mu)3. Polymerases α, δ and ε (alpha, delta, and epsilon)3. Polymerases η, ι and κ (eta, iota, and kappa)3. Polymerases Rev. 1 and ζ (zeta)3. Telomerase. 3. 2. Polymerases γ and θ (gamma and theta)3. Polymerase ν (nu)3. Reverse transcriptase. See also. 5References. Further reading. 7External links. History[edit]In 1. Arthur Kornberg and colleagues discovered DNA polymerase I (Pol I), in Escherichia coli. They described the DNA replication process by which DNA polymerase copies the base sequence of a template DNA strand. Kornberg was later awarded the Nobel Prize in Physiology or Medicine in 1. DNA polymerase II was also discovered by Thomas Kornberg (the son of Arthur Kornberg)[8] and Malcolm E. Gefter in 1. 97. 0 while further elucidating the role of Pol I in E. DNA replication.[9]Function[edit]. DNA polymerase moves along the old strand in the 3'- 5' direction, creating a new strand having a 5'- 3' direction. DNA polymerase with proofreading ability. The main function of DNA polymerase is to synthesize DNA from deoxyribonucleotides, the building blocks of DNA. The DNA copies are created by the pairing of nucleotides to bases present on each strand of the original DNA molecule. This pairing always occurs in specific combinations, with cytosine along with guanine, and thymine along with adenine, forming two separate pairs, respectively. By contrast, RNA polymerases synthesize RNA from ribonucleotides from either RNA or DNA. When synthesizing new DNA, DNA polymerase can add free nucleotides only to the 3' end of the newly forming strand. This results in elongation of the newly forming strand in a 5'- 3' direction. No known DNA polymerase is able to begin a new chain (de novo); it can only add a nucleotide onto a pre- existing 3'- OH group, and therefore needs a primer at which it can add the first nucleotide. Primers consist of RNA or DNA bases (or both). In DNA replication, the first two bases are always RNA, and are synthesized by another enzyme called primase. Enzymes, helicase and topoisomerase II, are required to unwind DNA from a double- strand structure to a single- strand structure to facilitate replication of each strand consistent with the semiconservative model of DNA replication. It is important to note that the directionality of the newly forming strand (the daughter strand) is opposite to the direction in which DNA polymerase moves along the template strand. Since DNA polymerase requires a free 3' OH group for initiation of synthesis, it can synthesize in only one direction by extending the 3' end of the preexisting nucleotide chain. Hence, DNA polymerase moves along the template strand in a 3'- 5' direction, and the daughter strand is formed in a 5'- 3' direction. This difference enables the resultant double- strand DNA formed to be composed of two DNA strands that are antiparallel to each other. The function of DNA polymerase is not quite perfect, with the enzyme making about one mistake for every billion base pairs copied. Error correction is a property of some, but not all DNA polymerases. This process corrects mistakes in newly synthesized DNA. When an incorrect base pair is recognized, DNA polymerase moves backwards by one base pair of DNA. The 3'- 5' exonuclease activity of the enzyme allows the incorrect base pair to be excised (this activity is known as proofreading). Following base excision, the polymerase can re- insert the correct base and replication can continue forwards. This preserves the integrity of the original DNA strand that is passed onto the daughter cells. Fidelity is very important in DNA replication. Mismatches in DNA base pairing can potentially result in dysfunctional proteins and could lead to cancer. Many DNA polymerase contain an exonuclease domain, which acts in detecting base pair mismatches and further performs in the removal of the incorrect nucleotide to be replaced by the correct one.[1. The shape and the interactions accommodating the Watson and Crick base pair are what primarily contribute to the detection or error. Hydrogen bonds play a key role in base pair binding and interaction. The loss of an interaction, which occurs at a mismatch, is said to trigger a shift in the balance, for the binding of the template- primer, from the polymerase, to the exonuclease domain. In addition, an incorporation of a wrong nucleotide causes a retard in DNA polymerization. This delay gives time for the DNA to be switched from the polymerase site to the exonuclease site. Different conformational changes and loss of interaction occur at different mismatches. In a purine: pyrimidine mismatch there is a displacement of the pyrimidine towards the major groove and the purine towards the minor groove. Relative to the shape of DNA polymerase’s binding pocket, steric clashes occur between the purine and residues in the minor groove, and important van der Waals and electrostatic interactions are lost by the pyrimidine.[1. Pyrimidine: pyrimidine and purine: purine mismatches present less notable changes since the bases are displaced towards the major groove, and less steric hindrance is experienced. However, although the different mismatches result in different steric properties, DNA polymerase is still able to detect and differentiate them so uniformly and maintain fidelity in DNA replication.[1. Structure[edit]The known DNA polymerases have highly conserved structure, which means that their overall catalytic subunits vary very little from species to species, independent of their domain structures. Conserved structures usually indicate important, irreplaceable functions of the cell, the maintenance of which provides evolutionary advantages. The shape can be described as resembling a right hand with thumb, finger, and palm domains. The palm domain appears to function in catalyzing the transfer of phosphoryl groups in the phosphoryl transfer reaction. DNA is bound to the palm when the enzyme is active. This reaction is believed to be catalyzed by a two- metal- ion mechanism. The finger domain functions to bind the nucleoside triphosphates with the template base. The thumb domain plays a potential role in the processivity, translocation, and positioning of the DNA.[1. Processivity[edit]DNA polymerase’s rapid catalysis is due to its processive nature. Processivity is a characteristic of enzymes that function on polymeric substrates. In the case of DNA polymerase, the degree of processivity refers to the average number of nucleotides added each time the enzyme binds a template. The average DNA polymerase requires about one second locating and binding a primer/template junction. Once it is bound, a nonprocessive DNA polymerase adds nucleotides at a rate of one nucleotide per second.[1. Processive DNA polymerases, however, add multiple nucleotides per second, drastically increasing the rate of DNA synthesis. The degree of processivity is directly proportional to the rate of DNA synthesis. The rate of DNA synthesis in a living cell was first determined as the rate of phage T4 DNA elongation in phage infected E. During the period of exponential DNA increase at 3. C, the rate was 7. Polymerase chain reaction - Wikipedia. A strip of eight PCR tubes, each containing a 1. Polymerase chain reaction (PCR) is a technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. It is an easy, cheap, and reliable way to repeatedly replicate a focused segment of DNA, a concept which is applicable to numerous fields in modern biology and related sciences.[1]Developed in 1. Kary Mullis,[2][3] PCR is now a common and often indispensable technique used in clinical and research laboratories for a broad variety of applications.[4][5] These include DNA cloning for sequencing, gene cloning and manipulation, gene mutagenesis; construction of DNA- based phylogenies, or functional analysis of genes; diagnosis and monitoring of hereditary diseases; amplification of ancient DNA; [6] analysis of genetic fingerprints for DNA profiling (for example, in forensic science and parentage testing); and detection of pathogens in nucleic acid tests for the diagnosis of infectious diseases. In 1. 99. 3, Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for his work on PCR.[7]The vast majority of PCR methods rely on thermal cycling, which involves exposing the reactants to cycles of repeated heating and cooling, permitting different temperature- dependent reactions—specifically, DNA melting and enzyme- driven DNA replication—to quickly proceed many times in sequence. Primers (short DNA fragments) containing sequences complementary to the target region, along with a DNA polymerase, after which the method is named, enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the original DNA template is exponentially amplified. The simplicity of the basic principle underlying PCR means it can be extensively modified to perform a wide array of genetic manipulations. PCR is not generally considered to be a recombinant DNA method, as it does not involve cutting and pasting DNA, only amplification of existing sequences. Almost all PCR applications employ a heat- stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the thermophilic bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from free nucleotides, the building blocks of DNA, by using single- stranded DNA as a template and DNA oligonucleotides (the primers mentioned above) to initiate DNA synthesis. In the first step, the two strands of the DNA double helix are physically separated at a high temperature in a process called DNA melting. In the second step, the temperature is lowered and the two DNA strands become templates for DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to sequence around the DNA region targeted for amplification under specific thermal cycling conditions. Principles[edit]PCR amplifies a specific region of a DNA strand (the DNA target). Most PCR methods amplify DNA fragments of between 0. The amount of amplified product is determined by the available substrates in the reaction, which become limiting as the reaction progresses.[9]A basic PCR set- up requires several components and reagents,[1. DNA template that contains the DNA target region to amplifya DNA polymerase, an enzyme that polymerizes new DNA strands; heat- resistant Taq polymerase is especially common,[1. DNA denaturation processtwo DNA primers that are complementary to the 3' (three prime) ends of each of the sense and anti- sense strands of the DNA target (DNA polymerase can only bind to and elongate from a double- stranded region of DNA; without primers there is no double- stranded initiation site at which the polymerase can bind); [1] specific primers that are complementary to the DNA target region are selected beforehand, and are often custom- made in a laboratory or purchased from commercial biochemical suppliersdeoxynucleoside triphosphates, or d. NTPs (sometimes called "deoxynucleotide triphosphates"; nucleotides containing triphosphate groups), the building blocks from which the DNA polymerase synthesizes a new DNA stranda buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA polymerasebivalentcations, typically magnesium (Mg) or manganese (Mn) ions; Mg. Mn. 2+ can be used for PCR- mediated DNA mutagenesis, as a higher Mn. DNA synthesis[1. 2]monovalent cations, typically potassium (K) ions. The reaction is commonly carried out in a volume of 1. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (see below). Many modern thermal cyclers make use of the Peltier effect, which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin- walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermal cyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube. Procedure[edit]Typically, PCR consists of a series of 2. The cycling is often preceded by a single temperature step at a very high temperature (> 9. C (1. 94 °F)), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters, including the enzyme used for DNA synthesis, the concentration of bivalent ions and d. NTPs in the reaction, and the melting temperature (Tm) of the primers.[1. The individual steps common to most PCR methods are as follows: Initialization: This step is only required for DNA polymerases that require heat activation by hot- start PCR.[1. It consists of heating the reaction chamber to a temperature of 9. C (2. 01–2. 05 °F), or 9. C (2. 08 °F) if extremely thermostable polymerases are used, which is then held for 1–1. Denaturation: This step is the first regular cycling event and consists of heating the reaction chamber to 9. C (2. 01–2. 08 °F) for 2. This causes DNA melting, or denaturation, of the double- stranded DNA template by breaking the hydrogen bonds between complementary bases, yielding two single- stranded DNA molecules. Annealing: In the next step, the reaction temperature is lowered to 5. C (1. 22–1. 49 °F) for 2. DNA templates. Two different primers are typically included in the reaction mixture: one for each of the two single- stranded complements containing the target region. The primers are single- stranded sequences themselves, but are much shorter than the length of the target region, complementing only very short sequences at the 3' end of each strand. It is critical to determine a proper temperature for the annealing step because efficiency and specificity are strongly affected by the annealing temperature. This temperature must be low enough to allow for hybridization of the primer to the strand, but high enough for the hybridization to be specific, i. If the temperature is too low, the primer may bind imperfectly. If it is too high, the primer may not bind at all. A typical annealing temperature is about 3–5 °C below the Tm of the primers used. Stable hydrogen bonds between complementary bases are formed only when the primer sequence very closely matches the template sequence. During this step, the polymerase binds to the primer- template hybrid and begins DNA formation. Extension/elongation: The temperature at this step depends on the DNA polymerase used; the optimum activity temperature for Taq polymerase is approximately 7. C (1. 67–1. 76 °F),[1. C (1. 62 °F) is commonly used with this enzyme. In this step, the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding free d. NTPs from the reaction mixture that are complementary to the template in the 5'- to- 3' direction, condensing the 5'- phosphate group of the d. NTPs with the 3'- hydroxy group at the end of the nascent (elongating) DNA strand. The precise time required for elongation depends both on the DNA polymerase used and on the length of the DNA target region to amplify.
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