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    DNA Polymerase—Four Key Characteristics for PCR

    Learn about DNA polymerase attributes important for successful PCR results.

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    DNA Polymerase–Four Key Characteristics for PCR

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    DNA polymerase is an essential component for PCR due to its key role in synthesizing new DNA strands. Consequently, understanding the characteristics of this enzyme and the subsequent development of advanced DNA polymerases is critical for adapting the power of PCR for a wide range of biological applications. Since the use of DNA polymerase in early PCR protocols, significant improvements have been made specifically in the specificity, thermostability, fidelity, and processivity of PCR enzymes. These properties of DNA polymerases have been modulated in combination to enhance PCR as described in the sections below.

    On this page

    Specificity Thermostability Fidelity Processivity

    This video explains enzyme properties such as thermostability, processivity, fidelity, and specificity in improving PCR results.

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    Specificity

    Nonspecific amplification is one of the major hurdles in PCR since it can drastically impact yield and sensitivity of target amplification, thereby compromising interpretation of results and the success of downstream applications. DNA polymerases often extend misprimed targets and primer-dimers, which are common sources of nonspecific amplification. One way to reduce nonspecific amplification is to set up PCR on ice. This helps keep the activity of the DNA polymerase low, but synthesis of undesirable products may still occur before the start of PCR. Another solution is to delay adding the DNA polymerase until the annealing step of the first cycle. This technique is termed “hot start” since amplification can start only after the initial denaturation step above 90°C.

    Learn about hot-start PCR and its benefits for your PCR applications. Discover how you can reduce nonspecific amplification and increase yield in PCR.

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    Although effective for improving specificity, the manual hot-start procedure is laborious and increases the risk of sample contamination and poor reproducibility. In 1994, DNA polymerases with a true hot-start property were developed [1,2], where specific antibodies are bound to the polymerases to inhibit them at room temperature during the reaction setup. During the initial high-temperature denaturation step (e.g., >90°C), the bound antibodies are degraded, activating the DNA polymerases (Figure 1).

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    Figure 1. Antibody-based hot-start DNA polymerase and its activation in PCR to enhance specificity.

    The denaturation step also separates misprimed targets and primer-dimers that may have formed during the reaction setup, thereby preventing their amplification by DNA polymerases in subsequent annealing and extension steps. In this manner, hot-start DNA polymerases reduce nonspecific amplification, increase yields, and allow convenient room temperature setup for high-throughput applications (Figures 2–4). (App note: High-throughput PCR).

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    Figure 2. PCR results from non–hot-start vs. hot-start DNA polymerases. Note the improved yields of the desired amplicon and lack of nonspecific amplification with a hot-start DNA polymerase.

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    Figure 3. Suitability of hot-start DNA polymerase for room-temperature reaction setup for high-throughput applications. PCR reactions were prepared and incubated at room temperature for 0, 24, and 72 hr before loading into a thermal cycler. Highly specific amplification of a 2 kb fragment from human gDNA was observed even 72 hr after room-temperature setup, demonstrating the power of hot-start DNA polymerases for large-scale experiments.

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    Figure 4. Comparison of polymerase activity: (A) a true “hot-start” DNA polymerase vs. (B) a “warm-start” DNA polymerase. Polymerase activity was measured at 60°C (constant) for 60 minutes. In heat-activation tests (blue curves), polymerases were heat-treated at 94°C for 2 minutes to dissociate the antibodies from the polymerases. Without heat activation (red curves), the true hot-start DNA polymerase showed no detectable activity, whereas the warm-start enzyme displayed activation at 60°C, making it unsuitable for hot-start applications.

    As alternatives to antibodies, hot-start attributes can be also achieved by heat-labile chemical modifications of the enzyme’s active site, as well as by using small molecules such as aptamers to shorten the activation time. Regardless of the choice of hot-start technologies, it is crucial that the DNA polymerase’s activity be efficiently blocked under unheated conditions to ensure specificity (Figure 4).

    Source : www.thermofisher.com

    Polymerase chain reaction (PCR) (article)

    A technique used to amplify, or make many copies of, a specific target region of DNA.

    Biotechnology

    Polymerase chain reaction (PCR)

    A technique used to amplify, or make many copies of, a specific target region of DNA.

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    Key points:

    Polymerase chain reaction, or PCR, is a technique to make many copies of a specific DNA region in vitro (in a test tube rather than an organism).

    PCR relies on a thermostable DNA polymerase, Taq polymerase, and requires DNA primers designed specifically for the DNA region of interest.

    In PCR, the reaction is repeatedly cycled through a series of temperature changes, which allow many copies of the target region to be produced.

    PCR has many research and practical applications. It is routinely used in DNA cloning, medical diagnostics, and forensic analysis of DNA.

    What is PCR?

    Polymerase chain reaction (PCR) is a common laboratory technique used to make many copies (millions or billions!) of a particular region of DNA. This DNA region can be anything the experimenter is interested in. For example, it might be a gene whose function a researcher wants to understand, or a genetic marker used by forensic scientists to match crime scene DNA with suspects.

    Typically, the goal of PCR is to make enough of the target DNA region that it can be analyzed or used in some other way. For instance, DNA amplified by PCR may be sent for sequencing, visualized by gel electrophoresis, or cloned into a plasmid for further experiments.

    PCR is used in many areas of biology and medicine, including molecular biology research, medical diagnostics, and even some branches of ecology.

    Taq polymerase

    Like DNA replication in an organism, PCR requires a DNA polymerase enzyme that makes new strands of DNA, using existing strands as templates. The DNA polymerase typically used in PCR is called Taq polymerase, after the heat-tolerant bacterium from which it was isolated (Thermus aquaticus).

    T. aquaticus lives in hot springs and hydrothermal vents. Its DNA polymerase is very heat-stable and is most active around

    70 °\text C 70°C

    70, °, start text, C, end text

    (a temperature at which a human or E. coli DNA polymerase would be nonfunctional). This heat-stability makes Taq polymerase ideal for PCR. As we'll see, high temperature is used repeatedly in PCR to denature the template DNA, or separate its strands.

    PCR primers

    Like other DNA polymerases, Taq polymerase can only make DNA if it's given a primer, a short sequence of nucleotides that provides a starting point for DNA synthesis. In a PCR reaction, the experimenter determines the region of DNA that will be copied, or amplified, by the primers she or he chooses.

    PCR primers are short pieces of single-stranded DNA, usually around

    20 20 20

    nucleotides in length. Two primers are used in each PCR reaction, and they are designed so that they flank the target region (region that should be copied). That is, they are given sequences that will make them bind to opposite strands of the template DNA, just at the edges of the region to be copied. The primers bind to the template by complementary base pairing.

    Template DNA:

    5' TATCAGATCCATGGAGT...GAGTACTAGTCCTATGAGT 3' 3' ATAGTCTAGGTACCTCA...CTCATGATCAGGATACTCA 5'

    Primer 1: 5' CAGATCCATGG 3' Primer 2:

    When the primers are bound to the template, they can be extended by the polymerase, and the region that lies between them will get copied.

    [More detailed diagram showing DNA and primer directionality]

    The steps of PCR

    The key ingredients of a PCR reaction are Taq polymerase, primers, template DNA, and nucleotides (DNA building blocks). The ingredients are assembled in a tube, along with cofactors needed by the enzyme, and are put through repeated cycles of heating and cooling that allow DNA to be synthesized.

    The basic steps are:

    Denaturation (

    96 °\text C 96°C

    96, °, start text, C, end text

    ): Heat the reaction strongly to separate, or denature, the DNA strands. This provides single-stranded template for the next step.

    Annealing (

    55 55 55 - 65 65 65 °\text C °C

    °, start text, C, end text

    ): Cool the reaction so the primers can bind to their complementary sequences on the single-stranded template DNA.

    Extension (

    72 °\text C 72°C

    72, °, start text, C, end text

    ): Raise the reaction temperatures so Taq polymerase extends the primers, synthesizing new strands of DNA.

    This cycle repeats 25 25 25 - 35 35 35

    times in a typical PCR reaction, which generally takes

    2 2 2 - 4 4 4

    hours, depending on the length of the DNA region being copied. If the reaction is efficient (works well), the target region can go from just one or a few copies to billions.

    Source : www.khanacademy.org

    Polymerase Chain Reaction (PCR)

    Polymerase Chain Reaction (PCR)

    Polymerase Chain Reaction (PCR) Introduction

    PCR (Polymerase Chain Reaction)

    is a revolutionary method developed by Kary Mullis in the 1980s. PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand. Because DNA polymerase can add a nucleotide only onto a preexisting 3'-OH group, it needs a primer to which it can add the first nucleotide. This requirement makes it possible to delineate a specific region of template sequence that the researcher wants to amplify. At the end of the PCR reaction, the specific sequence will be accumulated in billions of copies (amplicons).

    How It Works

    Components of PCR

    DNA template

    - the sample DNA that contains the target sequence. At the beginning of the reaction, high temperature is applied to the original double-stranded DNA molecule to separate the strands from each other.

    DNA polymerase

    - a type of enzyme that synthesizes new strands of DNA complementary to the target sequence. The first and most commonly used of these enzymes isTaqDNA polymerase (fromThermis aquaticus), whereasPfuDNA polymerase (fromPyrococcus furiosus) is used widely because of its higher fidelity when copying DNA. Although these enzymes are subtly different, they both have two capabilities that make them suitable for PCR: 1) they can generate new strands of DNA using a DNA template and primers, and 2) they are heat resistant.

    Primers

    - short pieces of single-stranded DNA that are complementary to the target sequence. The polymerase begins synthesizing new DNA from the end of the primer.

    Nucleotides (dNTPs or deoxynucleotide triphosphates)

    - single units of the bases A, T, G, and C, which are essentially "building blocks" for new DNA strands.

    RT-PCR

    (Reverse Transcription PCR) is PCR preceded with conversion of sample RNA into cDNA with enzyme

    reverse transcriptase

    .

    Limitations of PCR and RT-PCR

    The PCR reaction starts to generate copies of the target sequence exponentially. Only during the exponential phase of the PCR reaction is it possible to extrapolate back to determine the starting quantity of the target sequence contained in the sample. Because of inhibitors of the polymerase reaction found in the sample, reagent limitation, accumulation of pyrophosphate molecules, and self-annealing of the accumulating product, the PCR reaction eventually ceases to amplify target sequence at an exponential rate and a "plateau effect" occurs, making the end point quantification of PCR products unreliable. This is the attribute of PCR that makesReal-Time Quantitative RT-PCRso necessary.

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    "gene expression"[application] 0

    "primer set"[probe type] AND "mus musculus"[organism] 0

    TaqMan[probe type] AND "wet lab success"[validation] 0

    Resources

    » "Polymerase Chain Reaction"[MAJR]

    Note: [MAJR] is a Medical Subject Heading (MeSH) tag for Major Heading. The tag is used to limit the search to articles for which major subjects are represented by terms included in the NLM MeSH database.

    Source : www.ncbi.nlm.nih.gov

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