Molecular Biology Electrophoresis Quiz 3
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Questions and Answers

Which of the following is NOT a characteristic of an ideal electrophoretic carrier?

  • Allows for adjustment of pore size by changing carrier concentration
  • Reacts with the protein or DNA being separated (correct)
  • Contributes to better separation of macromolecules
  • Provides stability to the electrolyte

    • What is the primary advantage of using a porous carrier in electrophoresis?

  • It enhances the separation effect by fractionating macromolecules based on molecular sieving (correct)
  • It stabilizes the electrolyte, preventing fluctuations in pH
  • It facilitates the migration of larger molecules towards the anode
  • It allows for the selective separation of charged molecules

    • How does increasing the concentration of agarose in a gel affect its properties?

  • It increases the stability of the gel, making it less prone to breakage
  • It decreases the pore size, acting as a more effective sieve for smaller molecules (correct)
  • It increases the pore size, allowing larger molecules to migrate faster
  • It decreases the viscosity of the gel, leading to faster migration of all molecules

    • Which of the following statements is TRUE regarding the use of electrophoresis in DNA separation?

    <p>Smaller DNA fragments migrate faster than larger fragments in an agarose gel (B)</p> Signup and view all the answers

    What is the primary reason for using different concentrations of agarose in gel electrophoresis?

    <p>To achieve different pore sizes for separating molecules of varying dimensions (D)</p> Signup and view all the answers

    What is the optimal concentration range for Mg2+ ions in most polymerase reactions?

    <p>1.5 to 2.0 mM (C)</p> Signup and view all the answers

    What is the consequence of using an unequal amount of dNTPs in a PCR reaction?

    <p>Reduced amplification efficiency (D)</p> Signup and view all the answers

    Why does too high a concentration of template DNA increase the amount of non-specific PCR products?

    <p>It increases the likelihood of mispriming (A)</p> Signup and view all the answers

    What is the optimal concentration range for primers in a PCR reaction?

    <p>0.05 - 1 μM (C)</p> Signup and view all the answers

    Which of the following is NOT a characteristic of Taq polymerase?

    <p>3'-5' exonuclease activity (A)</p> Signup and view all the answers

    What is the optimal concentration range for dNTPs in a PCR reaction?

    <p>200 - 400 μM (D)</p> Signup and view all the answers

    Why does too low an annealing temperature result in the appearance of non-specific PCR products?

    <p>Primers bind to unintended sequences in the template (B)</p> Signup and view all the answers

    What is the typical concentration of Taq polymerase used in a PCR reaction?

    <p>1 U per reaction (A)</p> Signup and view all the answers

    Which of the following factors can affect the efficiency of a PCR reaction?

    <p>All of the above (D)</p> Signup and view all the answers

    What does the term 'thermostability' refer to in relation to Taq polymerase?

    <p>The ability of the enzyme to withstand high temperatures (C)</p> Signup and view all the answers

    Which of the following statements accurately describes the effect of too high an annealing temperature during PCR?

    <p>It prevents primer binding to the template sequence altogether, resulting in no PCR product. (D)</p> Signup and view all the answers

    What is the primary reason for using Gradient PCR?

    <p>To determine the optimal annealing temperature for a specific primer pair and template sequence. (A)</p> Signup and view all the answers

    What is the primary function of the primer forward in PCR?

    <p>To anneal to the template sequence at a specific location and initiate DNA synthesis in the 5’-to-3’ direction. (C)</p> Signup and view all the answers

    What is the function of the reaction buffer component in PCR?

    <p>It provides the optimal pH and salt concentration required for DNA polymerase activity. (A)</p> Signup and view all the answers

    What is the significance of 100% primer matching to the template sequence at the optimal annealing temperature during PCR?

    <p>It ensures that the primer binds to the correct target sequence, leading to the amplification of the specific DNA fragment. (C)</p> Signup and view all the answers

    What is the consequence of having an annealing temperature too low during PCR?

    <p>It leads to the formation of non-specific products, as primers can bind to non-target sequences due to mismatches. (D)</p> Signup and view all the answers

    Which of the following factors contributes to the specificity of PCR amplification?

    <p>The use of primers that are specific to the target sequence. (A)</p> Signup and view all the answers

    The presence of non-specific products in a PCR reaction suggests which of the following?

    <p>The annealing temperature was too low, allowing primers to bind to non-target sequences. (C)</p> Signup and view all the answers

    What happens to PCR efficiency if the annealing temperature is too high?

    <p>It decreases, reducing the yield of the desired PCR product. (D)</p> Signup and view all the answers

    In the context of PCR, what is the significance of the 3’ end of the primer?

    <p>It provides a binding site for the DNA polymerase to initiate DNA synthesis. (D)</p> Signup and view all the answers

    Which of the following best describes the purpose of PCR reactions?

    <p>DNA amplification and copying specific genes (A)</p> Signup and view all the answers

    What is the primary function of the thermal cycler in a PCR reaction?

    <p>To rapidly change temperatures during the reaction stages (A)</p> Signup and view all the answers

    What occurs specifically during the denaturation stage of PCR?

    <p>Hydrogen bonds between DNA strands are broken (C)</p> Signup and view all the answers

    During which step of the PCR cycle do primers bind to the target DNA?

    <p>Annealing (C)</p> Signup and view all the answers

    What is the role of the Peltier element in a thermal cycler?

    <p>To enable rapid temperature transitions (C)</p> Signup and view all the answers

    How long is the denaturation step typically performed during PCR?

    <p>30 seconds to 5 minutes (A)</p> Signup and view all the answers

    What is typically the optimal temperature for the elongation phase in PCR?

    <p>72-75°C (D)</p> Signup and view all the answers

    What is the theoretical maximum rate of nucleotide addition during DNA synthesis?

    <p>1000 nt/min (B)</p> Signup and view all the answers

    How many cycles of PCR should be performed if the initial amount of template DNA is fewer than 10 copies?

    <p>40 cycles (C)</p> Signup and view all the answers

    What is the theoretical yield of DNA after 6 cycles of PCR?

    <p>64 double-stranded molecules (A)</p> Signup and view all the answers

    Which component is NOT required for a PCR reaction?

    <p>Antibodies (D)</p> Signup and view all the answers

    Which factor is NOT mentioned as influencing PCR efficiency?

    <p>Visibility of DNA (B)</p> Signup and view all the answers

    What is the correct direction of DNA strand extension by DNA polymerase?

    <p>5' to 3' (A)</p> Signup and view all the answers

    If 20 cycles of PCR are conducted, how many double-stranded DNA molecules are theoretically generated?

    <p>1,048,576 (D)</p> Signup and view all the answers

    Which of the following ensures the stability of DNA polymerase at high temperatures during PCR?

    <p>Buffer containing Mg2+ (D)</p> Signup and view all the answers

    What is the number of double-stranded DNA chains produced after 4 cycles of PCR?

    <p>16 (A)</p> Signup and view all the answers

    Flashcards

    Nucleotide Addition Rate

    The theoretical rate at which nucleotides are added during DNA synthesis, which is 1000 nucleotides per minute.

    Direction of DNA Synthesis

    DNA polymerase extends the primer in the 5' to 3' direction when copying the template strand.

    PCR Cycle Count

    The number of cycles in a PCR depends on the template DNA amount; less than 10 copies require about 40 cycles.

    Yield After PCR Cycles

    The theoretical yield after "n" cycles of PCR is 2^n double-stranded DNA molecules generated.

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    Exponential Amplification

    With each PCR cycle, the number of DNA chains doubles, leading to exponential growth.

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    Components of PCR Mixture

    Essential components for a PCR reaction include template DNA, primers, DNA polymerase, dNTPs, buffer, and water.

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    Annealing Temperature

    The temperature at which primers bind to the template DNA during PCR, crucial for efficiency.

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    MgCl2 Role

    Magnesium chloride helps stabilize the DNA polymerase and is vital for PCR efficiency.

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    dNTP Concentration

    Concentration of deoxyribonucleotide triphosphates (dATP, dTTP, dCTP, dGTP) affects PCR efficiency.

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    Electrophoretic Carrier

    Material used to stabilize electrolyte and enhance macromolecule separation during electrophoresis.

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    Gel Electrophoresis

    A technique where DNA is separated into bands by size in a gel matrix during an electric current.

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    Agarose

    A natural polysaccharide used to create gels for electrophoresis, derived from red seaweed, soluble in water.

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    Pore Size Adjustment

    The modification of the gel's pore size by altering agarose concentration to select molecule separation range.

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    Molecular Sieving

    Separation method based on size, utilized in porous carriers to fractionate macromolecules.

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    Polymerase Chain Reaction (PCR)

    A method for amplifying DNA in vitro by mimicking natural replication.

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    DNA amplification

    The process of creating multiple copies of a specific DNA sequence.

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    Thermal cycler

    A specialized machine that controls temperature cycles during PCR.

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    Denaturation

    The first step in PCR where DNA is heated to separate its strands.

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    Annealing

    The stage in PCR where primers bind to the DNA template.

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    Extension (Elongation)

    The process where new DNA strands are synthesized from the primers.

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    Kary Mullis

    Scientist who developed PCR and won a Nobel Prize for it.

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    Polymerase (Taq) concentration

    The amount of Taq polymerase enzyme used in PCR.

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    Primer extension temperature

    The temperature at which DNA primers elongate during PCR.

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    Reaction buffer

    A solution that provides the optimal environment for PCR reactions.

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    Optimal annealing temperature

    The perfect temperature where primers bind to DNA templates effectively.

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    High annealing temperature

    A temperature that prevents primer binding, reducing PCR efficiency.

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    Low annealing temperature

    A temperature that may cause less specific primer binding.

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    100% primer matching

    Complete alignment of primers with template DNA at optimal annealing temperature.

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    Gradient PCR

    A method used to optimize annealing temperatures by testing multiple values simultaneously.

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    Specific product

    The desired DNA fragment resulting from successful PCR amplification.

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    Non-specific product

    Undesired fragments formed from improper binding of primers during PCR.

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    PCR Efficiency

    The effectiveness of PCR reactions to amplify DNA, usually below 100%.

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    Template DNA Range (plasmid)

    Optimal plasmid DNA template amount is 0.01 – 1 ng for PCR.

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    Template DNA Range (genomic)

    Optimal genomic DNA template amount is 0.1 – 1 µg for PCR.

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    Primer Concentration

    The optimal primer concentration in PCR is 0.2 - 0.3 μM.

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    Divalent Ions Requirement

    Essential for polymerase activity, typically Mg2+, sometimes Mn2+.

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    Optimal Mg2+ Concentration

    Mg2+ concentration should be 1.5 - 2.0 mM for optimal PCR performance.

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    dNTPs Requirement

    Equal amounts of dATP, dTTP, dCTP, and dGTP, with 200 - 400 μM being optimal.

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    Taq Polymerase

    Thermostable enzyme from Thermus aquaticus, used for synthesizing DNA in PCR.

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    Taq Polymerase Misincorporation Rate

    Taq has a misincorporation rate of 2x10-4 to 2x10-5, affecting accuracy.

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    Study Notes

    PCR Reaction Overview

    • Polymerase Chain Reaction (PCR) is a technique for selectively amplifying DNA sequences in vitro, mimicking in vivo DNA replication.
    • It can duplicate DNA sequences ranging from a few hundred to several thousand nucleotides in length.
    • PCR was developed in 1987 by scientists at Cetus Corporation.
    • Kary Mullis received the Nobel Prize in Chemistry in 1993 for his work on PCR.

    Purposes of PCR

    • DNA amplification for various applications.
    • Detecting specific DNA sequences within a sample
      • Gene expression analysis
      • Diagnostics, mutation detection
      • Pathogen identification
      • Forensic science.
    • Copying specific DNA sequences for
      • Genetic engineering
      • Gene cloning
      • Functional gene analysis
      • Organism modification

    Thermal Cycler

    • PCR reactions occur in specialized thermal cyclers.
    • These devices precisely control temperature changes of dozens of degrees within seconds.
    • Thermal cyclers program the temperature and duration of each reaction stage and the number of cycles.
    • The core component of a thermal cycler is an aluminum block coupled with a Peltier element.

    PCR Cycle Stages

    • PCR involves three primary stages repeated 30 to 40 times:
      • Denaturation: DNA heating (92-96°C) to separate the double helix into single strands.
        • Duration depends on template DNA type;
      • Annealing: Primers (short, single-stranded DNA segments complementary to specific DNA sequences ) bind to the complementary regions on the single-stranded DNA template (37-72°C).
        • 15-60 seconds;
      • Extension: DNA polymerase synthesizes new DNA by extending the primers (68-75°C).
        • Elongation rate varies depending on the polymerase type.

    PCR Cycle Thermal Profile

    • The presentation of PCR cycle thermal profile provides a specific graph showing temperatures and times for each stage of the PCR cycle.

    Number of DNA Amplification Cycles

    • The number of cycles in PCR depends on the initial amount of template DNA and the desired yield.
    • Fewer than 10 template DNA copies often require 40 cycles for optimal amplification.
    • Higher initial copy numbers require fewer cycles (25-35 cycles).

    Exponential DNA Amplification

    • The number of DNA copies doubles with each PCR cycle, an exponential amplification.
    • This graph demonstrates an escalating duplication of DNA molecules after each cycle

    Components of PCR Mixture

    • Template DNA: The DNA to be amplified.
    • Primers: Short, single-stranded DNA segments complementary to specific DNA regions.
    • DNA polymerase (e.g., Taq polymerase): Enzyme catalyzing DNA synthesis.
    • dNTPs (Deoxynucleotide triphosphates): Building blocks for new DNA strands.
    • Buffer: Maintains reaction conditions. (e.g., MgCl₂ and other ions)
    • Nuclease-free water: Solvent.

    PCR Efficiency Factors

    • Annealing temperature: Optimal primer binding conditions.
    • Primer concentration/template DNA concentration: Adequate amounts to assure efficiency.
    • MgCl₂ concentration: Influences DNA polymerase activity.
    • dNTP concentration: Crucial for DNA synthesis.
    • Polymerase (Taq) concentration: Enough to catalyze production.
    • Primer extension temperature: Appropriate for enzyme activity.
    • Reaction buffer: Consistent pH and ionic strength.

    PCR Reaction Optimization

    • PCR reactions typically don't achieve 100% efficiency.
    • Adjustments of reaction conditions (including annealing temperature, primer concentrations, and Mg2+ concentration) are required to optimize results.

    Template and Primers Concentration

    • Optimal template DNA concentrations to prevent non-specific results (too much DNA, or too little primers).
    • Primer concentrations have optimal ranges.

    Divalent Ions (Mg²⁺)

    • Mg2+ ions are essential for DNA polymerase activity.
    • The Mg2+ concentration should exceed the number of phosphate groups in the reagents to which it binds.
    • The optimal concentration is typically 1.5-2.0 mM.

    Deoxynucleotides (dNTPs)

    • Equal amounts of dNTPs are essential for accurate DNA synthesis;
    • Optimal dNTP concentration is between 200 and 400 μM;

    Taq Polymerase

    • Thermostable DNA polymerase enzyme, active in PCR's high-temperature ranges.
    • 3'-5' exonuclease activity (removing incorrectly added nucleotides) varies depending on whether or not it is present.
    • Concentration in the reaction is 0.04 – 0.1 U/µl.

    Elongation Temperature

    • Optimal temperature for DNA polymerase activity is usually 72°C (with different ranges depending on the enzyme).
    • Temperature can affect the elongation rate. Lower temperatures may be required for accuracy in certain reactions.

    Reaction Buffer

    • Specific to a particular polymerase; it stabilizes the pH of the reaction mixture.
    • Standard buffer contains potassium chloride (KCl) maintaining appropriate pH and ionic strength.

    Reaction Mixture Additives

    • These additives can include Bovine Serum Albumin (BSA), ammonium sulfate, and Triton (a chemical detergent). These additives can stabilize the DNA polymerase.

    PCR Risks

    • False positive: Post-amplification contaminants and positive control contamination.
    • False negative: Reaction inhibitors, improper sample volume, inaccurate protocol, and poor quality nucleic acid.

    Necessary Control Reactions

    • Positive control: A known DNA template (known primer binding sequence) establishes a success marker.
    • Negative control: PCR mix with water instead of DNA helps rule out external contamination.

    PCR Varieties

    • End-point PCR: Amplified product analysis (e.g. gel electrophoresis) determines the quality but not the quantity.
    • Multiplex PCR: Simultaneous amplification of multiple regions across the genome.
    • Real-Time PCR: Quantitative measurements (fluorescence) provide real-time data to detect and measure amplified DNA.

    End-point PCR vs. Real-Time PCR

    • End-point PCR is simple and qualitative.
    • Real-time PCR obtains quantitative results through fluorescence during the reaction.

    Reverse Transcriptase PCR (RT-PCR)

    • Used to study mRNA, converting it to cDNA prior to PCR.
    • Reverse transcriptase synthesizes cDNA from an RNA template.
    • This is important for analysis of gene expressions.

    DNA Electrophoresis

    • Analytical technique to separate DNA (or protein) molecules based on size.
    • Electrophoresis with electrophoretic carriers provides higher stability, repeatability, and resolution.

    Electrophoresis Techniques

    • Zone electrophoresis: Separation occurs in an electrolyte solution with a stable pH.
    • Horizontal electrophoresis: Gels are positioned horizontally, commonly used to separate DNA or other large molecules.
    • Vertical electrophoresis: Used for protein separation.

    Electrophoresis Carriers

    • Porous carriers (e.g., agarose, polyacrylamide gels) separate molecules based on their size.
    • Agarose gels separate wider DNA range; polyacrylamide provides higher resolution and is good for small fragments or molecules.

    Agarose Gels

    • Agarose is derived from seaweed, forming gels at room temperature.
    • Pore sizes in agarose gels are controlled using differing concentrations.
    • Typical range of agarose concentration between 0.4% and 4.0%.

    Polyacrylamide Gels

    • Prepared with acrylamide monomers and cross-linking substances.
    • Pore sizes are adjusted to target molecules of differing sizes.

    Electrophoresis Chambers

    • Used to conduct electrophoresis, with subtypes based on placement of the electrophoretic carriers.
    • Vertical type: Used to separate proteins.
    • Horizontal: Used to separate DNA.
    • Capillary: Another variation to separate other kinds of molecules.

    Documentation

    • Polyacrylamide gel drying using filter paper and/or cellophane for preservation.
    • Agarose gel documentation is mainly through photographic record.

    Staining

    • Proteins and nucleic acids are often not visible without staining.
    • Staining techniques like Coomassie Brilliant Blue are used for protein staining, while Silver staining and fluorescent dyes (e.g., ethidium bromide) stain nucleic acids.

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    Description

    This quiz focuses on key concepts in molecular biology related to electrophoresis and PCR. Questions cover characteristics of electrophoretic carriers, gel concentration effects, and optimal conditions for PCR reactions. Test your knowledge on important techniques used for DNA separation and analysis.

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