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    BIO 123 Quiz 2 Results Flashcards

    Start studying BIO 123 Quiz 2 Results. Learn vocabulary, terms, and more with flashcards, games, and other study tools.

    BIO 123 Quiz 2 Results

    In E. coli, there is a mutation in a gene called dnaB that alters the helicase that normally acts at the origin of replication. Which of the following events would you expect to occur as a result of this mutation?

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    No replication fork will be formed.

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    In E. coli, what is the function of DNA polymerase III?

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    to add nucleotides to the 3' end of a growing DNA strand

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    Terms in this set (10)

    In E. coli, there is a mutation in a gene called dnaB that alters the helicase that normally acts at the origin of replication. Which of the following events would you expect to occur as a result of this mutation?

    No replication fork will be formed.

    In E. coli, what is the function of DNA polymerase III?

    to add nucleotides to the 3' end of a growing DNA strand

    Which of the following types of molecules help to hold the DNA strands apart while they are being replicated?

    single-strand DNA binding proteins

    What is the role of DNA ligase in the elongation of the lagging strand during DNA replication?

    It joins Okazaki fragments together.

    In the polymerization of DNA, a phosphodiester bond is formed between a phosphate group of the nucleotide being added and which of the following atoms or molecules of the last nucleotide in the polymer?

    the 3' OH

    In E. coli, to repair a thymine dimer by nucleotide excision repair, in which order do the necessary enzymes act?

    nuclease, DNA polymerase, DNA ligase

    Which of the following characteristics would you expect of a eukaryotic organism that lacks the enzyme telomerase?

    a reduction in chromosome length in gametes

    In E. coli, which enzyme catalyzes the elongation of a new DNA strand in the 5' → 3' direction?

    DNA polymerase III

    Which of the following characteristics of Taq polymerase make it useful in the PCR process?

    It is heat stable and can withstand the heating step of PCR.

    Which of the following correctly lists the processes in order for one cycle of polymerase chain reaction (PCR)?

    denature DNA; anneal primers; extend primers

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    Taq Polymerase

    Taq Polymerase

    The Taq polymerase enzymes utilized in these TaqMan assays (either Taq or Tth) possess a 5′ to 3′ exonuclease activity, and as a result, during replication of one of the DNA strands the enzyme will encounter the bound probe and cleave it, resulting in the release of the reporter dye into solution.

    From: Advances in Virus Research, 2003

    Related terms:

    EnzymeProteinDNARNAPhosphoproteinPolymerase Chain ReactionAlleleComplementary DNA

    View all Topics

    Laboratory Approaches in Molecular Pathology—The Polymerase Chain Reaction

    W.B. Coleman, G.J. Tsongalis, in Diagnostic Molecular Pathology, 2017

    Increasing PCR Specificity and Sensitivity

    Taq polymerase has substantial enzymatic activity at 37°C, although its optimal activity is expressed at a much higher temperature (approximately 72°C). This low-temperature polymerase activity is the basis for nonspecific amplification associated with mispriming events that occur during the initial phase of the PCR reaction. Extension can occur from oligodeoxynucleotide primers that anneal nonspecifically to template DNA before the first denaturation step at 93–95°C. Because of this, several modified polymerase enzymes have been created to avoid this nonspecific primer extension activity. One example of this is Platinum Taq Polymerase (from Invitrogen Life Technologies, http://www.thermofisher.com). By including a thermolabile inhibitor of Taq polymerase in the form of a monoclonal antibody, the enzyme does not become active until the inhibitor is heat inactivated. Hence, the Taq polymerase becomes active after the elevated temperature destroys the monoclonal antibody during the initial denaturation phase of the PCR reaction which results in release of the functional enzyme. The antibody-mediated inhibition of Taq polymerase allows for room temperature assembly of the PCR reaction mixture. Nonspecific amplification associated primer extension from mispriming events is eliminated or reduced by holding the Taq polymerase functionally inactive until the critical temperature is reached.

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    General Considerations

    Gregory A. Denomme PhD, ... Marion E. Reid PhD, in Molecular Protocols in Transfusion Medicine, 2000

    2.2.4 DNA Polymerases

    Taq DNA polymerase is the most common enzyme used for PCR amplification. This enzyme is extremely heat resistant with a half-life of 40 minutes at 95°C. At its optimal temperature (72°C), nucleotides are incorporated at a rate of 2–4 kilobases per minute. However, polymerases are active over a broad temperature range (Gelfand and White 1990). Therefore, if DNA, primers and Taq polymerase are mixed together, mis-priming and elongation can occur at room temperature (before the denaturation step). This will result in the amplification of non-specific targets that can be overcome by the use of a ‘hot-start’ PCR technique (Mullis 1991). Typically the technique involves either: (1) adding the polymerase during the initial denaturation step; (2) separating the enzyme from the other reagents using a paraffin layer (AmpliWax™; Roche Molecular Systems, Inc.); or (3) using a modified enzyme that is activated upon heating to 95°C for 15 minutes. In each instance, polymerase activity is prevented until the initial annealing stage.

    Taq polymerase does not have 3′–5′ proof-reading activity. However, it has a very low mis-incorporation rate; estimated at 1 per 10,000 bases. Proof-reading enzymes must be avoided when performing AS-PCR, since these enzymes will correct the deliberate mis-match necessary for genotyping.

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    The Polymerase Chain Reaction

    Cornel Mülhardt, E.W. Beese M.D., in Molecular Biology and Genomics, 2007

    4.3.11 Cycle Sequencing

    Taq polymerase has been employed for some time in sequencing. In contrast to the normal PCR, only a primer is used, so only linear growth (not exponential) is observed.

    Taq functions at higher temperatures than a classic DNA polymerase and, in part, even permits better sequencing results, because the GC-rich structures can be broken down better. Perhaps it is also the somewhat reduced pipetting or merely the tendency of the PCR lover to solve all problems with their PCR apparatus, which has helped to make cycle sequencing so popular. More on the topic of sequencing can be found in Chapter 8, Section 8.1.

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    Thermostable DNA Polymerases

    Richard D. Abramson, in PCR Strategies, 1995

    Taq DNA Polymerase

    Taq DNA polymerase is an 832-amino acid protein with an inferred molecular weight of 93,920 and a specific activity of 292,000 units/ mg; optimal polymerization activity is achieved at 75–80 ° C, with half-maximal activity at 60–70 ° C (Lawyer et al., 1993; see also Table 1). It's thermostability as measured as a half-life of activity is between 45 and 96 min at 95 ° C and 9 min at 97.5 ° C (Lawyer et al., 1993; Kong et al., 1993). Maximal enzymatic activity is achieved in a N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (Taps)-KOH (25 mM), pH 9.4 buffer containing 10–55 mM KCl and 2–3 mM MgCl2 (Lawyer et al., 1993). Under conditions of enzyme excess, the polymerase extends a primer at a maximum rate of 75 nucleotides per second at 70 ° C (Innis et al., 1988; Abramson et al., 1990). The enzyme is moderately processive, extending a primer an average of 50–60 nucleotides before it dissociates (King et al., 1993; Abramson et al., 1990). The fidelity of polymerase base substitution has been estimated to range between 3 × 10–4 and 3 × 10–6 errors per nucleotide polymerized, depending on reaction conditions (Eckert and Kunkel, 1992). In a standard PCR, amplification with Taq DNA polymerase is optimal in a Tris–HCl (10 mM), pH 8.3 buffer containing 50 mM KCl and 1.5 mM MgCl2, although MgCl2 concentration may need to be adjusted for any specific primer pair-template combination (Innis and Gelfand, 1990). Based on sequence homology to Escherichia coli DNA polymerase I, Taq DNA polymerase has been included in a family of DNA polymerases known as Family A (Braithwaite and Ito, 1993). Like E. coli DNA polymerase I, Taq DNA polymerase possesses an 5′ to 3′ exonuclease or "nicktranslation" activity (Longley et al., 1990; Holland et al., 1991; Lawyer et al., 1993). It does not, however, possess a 3′ to 5′ exonuclease or "proofreading" activity (Tindall and Kunkel, 1988; Lawyer et al., 1989).

    Source : www.sciencedirect.com

    Taq Polymerase is Preferred Enzyme for Polymerase Chain Reaction (PCR)

    Taq Polymerase may play an important role in PCR, but what exactly does it do? To learn more about Taq DNA Polymerase, click here!

    Taq Polymerase is Preferred Enzyme for Polymerase Chain Reaction (PCR)

    Posted by The Protein Man on Mar 17, 2020 2:30:00 PM

    A DNA Polymerase is a vital biological enzyme that is present in DNA replication. In the process, DNA copies into two daughter DNA molecules and synthesizes a new DNA strand from the existing strand by adding dNTPs to the growing DNA.

    The DNA Polymerase has two crucial roles in replication: 5’ to 3’ exonuclease polymerase activity and 3’ to 5’ exonuclease proofreading activity. As the polymerase binds to DNA, it adds nucleotide in the direction of 5’ to 3’.

    Unfortunately, because it disables at a higher temperature, DNA Polymerase is not suitable for a type of replication called Polymerase Chain Reaction (PCR). Taq DNA Polymerase, on the other hand, plays an essential role in PCR.

    What is Taq Polymerase?

    Taq DNA Polymerase is a highly thermostable recombinant DNA polymerase. It is named after Thermus aquaticus, the heat-tolerant bacterium from which it isolates itself. The molecular weight of the Taq Polymerase is 94kD, and it amplifies DNA up to 5kb with an elongation velocity of 0.9-1.2kb/min at 70-75°C.

    What is Polymerase Chain Reaction (PCR)?

    Polymerase Chain Reaction, or PCR, is a replication technique that produces numerous copies of a specific DNA region in vitro (in a test tube rather than an organism). PCR is utilized in various areas of biology and medicine, including molecular biology research, medical diagnostics, and ecology.

    PCR relies upon Taq Polymerase, a thermostable DNA polymerase. It also requires DNA primers that are designed specifically for the DNA region of interest, as well as template DNA and nucleotides (DNA building blocks).

    In a PCR reaction, you will assemble the key ingredients in a tube, along with cofactors the enzyme requires. The ingredients undergo repeated cycles of heating and cooling that ultimately synthesize DNA.

    There are also three basic steps you must follow to achieve PCR, including:

    Denaturation (96 °C): Heat the reaction strongly to separate, or denature, the DNA strands.Annealing (55-65 °C): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA.Extension (72 °C): Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA.

    The cycle repeats itself 25-35 times in a typical PCR reaction, which generally takes 2-4 hours, depending on the length of the DNA region being copied. If the reaction is efficient, the target region can go from just one or a few copies to billions.

    The Role of Taq Polymerase in PCR

    Due to its key role in synthesizing and amplifying new strands of DNA, Taq DNA Polymerase is essential to Polymerase Chain Reaction (PCR).

    Like other DNA polymerases, Taq Polymerase can only produce DNA if it has a primer, a short sequence of 20 nucleotides that provide a starting point for DNA synthesis. However, it is also a thermostable DNA polymerase that can work at higher temperatures.

    Taq DNA Polymerase is highly efficient, so it becomes fully functional as it reaches its optimum temperature. It also has a half-life of more than two hours (at a temperature of 92 °C), a high-amplification capacity, and the ability to add 150 nucleotides per second.

    Choose G-Biosciences for Your PCR Essentials

    At G-Biosciences, we offer a variety of kits and products for your PCR and Taq DNA Polymerase applications. To place your order, visit our website today.

    Topics: Molecular Biology

    Source : info.gbiosciences.com

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