Lab 10: PCR and Laboratory Instrumentation
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Questions and Answers

What is the primary function of thermostable DNA polymerase in PCR?

  • To anneal primers to the DNA template
  • To provide magnesium for DNA polymerase activity
  • To denature the DNA template
  • To synthesize new strands of DNA (correct)

    • What is the purpose of the initialization step in PCR?

  • To denature the DNA template
  • To anneal primers to the DNA template
  • To activate heat-labile DNA polymerases (correct)
  • To extend primers with nucleotides

    • What is the role of magnesium in PCR?

  • To act as a co-factor for DNA polymerase (correct)
  • To denature the DNA template
  • To provide a substrate for nucleotide extension
  • To activate DNA polymerase

    • What is the purpose of the denaturation step in PCR?

    <p>To denature the DNA template</p> Signup and view all the answers

    What is the typical number of temperature cycles in a PCR reaction?

    <p>25-40</p> Signup and view all the answers

    What is the name of the thermophilic bacterium from which Taq DNA polymerase was originally isolated?

    <p>Thermus aquaticus</p> Signup and view all the answers

    What is the purpose of the PCR buffer in PCR?

    <p>To create optimal conditions for Taq DNA polymerase activity</p> Signup and view all the answers

    What are dNTPs used for in PCR?

    <p>To provide a substrate for DNA polymerase activity</p> Signup and view all the answers

    What is the temperature range for the annealing step in PCR?

    <p>50-65°C</p> Signup and view all the answers

    What is the purpose of the final elongation step?

    <p>To ensure complete extension of remaining single-stranded DNA</p> Signup and view all the answers

    What is the function of the thermostable DNA polymerase during annealing?

    <p>To extend the primers as soon as they anneal to the template</p> Signup and view all the answers

    What is the optimal temperature for Taq polymerase activity?

    <p>72-80°C</p> Signup and view all the answers

    What is the direction of DNA synthesis during the extension step?

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

    What is the purpose of gel electrophoresis in PCR?

    <p>To detect the amplified product</p> Signup and view all the answers

    What happens to the single strands of the template DNA during the annealing step?

    <p>They remain single-stranded and do not reanneal</p> Signup and view all the answers

    What is the outcome if the target sequence is not present in the original DNA sample?

    <p>No band is visible on the gel</p> Signup and view all the answers

    What is the temperature in the denaturation step of a typical PCR thermal cycle?

    <p>94°C</p> Signup and view all the answers

    What is the primary application of PCR in forensic science?

    <p>Identification of criminal</p> Signup and view all the answers

    What is the purpose of the final elongation step in PCR?

    <p>To ensure complete extension of the DNA strands</p> Signup and view all the answers

    What is the advantage of PCR in detecting microorganisms?

    <p>It can detect microorganisms that are difficult to culture</p> Signup and view all the answers

    What is the purpose of PCR in medicine?

    <p>To detect pathogens</p> Signup and view all the answers

    What is the primary application of PCR in evolutionary studies?

    <p>Phylogenetic analysis</p> Signup and view all the answers

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

    <p>To heat and cool the reaction tubes to achieve the required temperatures</p> Signup and view all the answers

    What is the purpose of the forward and reverse primers in PCR?

    <p>To initiate DNA synthesis and specify the amplification to the target DNA segment</p> Signup and view all the answers

    What is the minimum amount of DNA required for PCR to work?

    <p>A single copy of the DNA sequence</p> Signup and view all the answers

    Who developed the Polymerase Chain Reaction (PCR) technique?

    <p>Kary Mullis</p> Signup and view all the answers

    What is the function of the template DNA in PCR?

    <p>To carry the DNA segment or target you wish to amplify</p> Signup and view all the answers

    What is the importance of free nuclease water in PCR?

    <p>It must be present for the reaction to work</p> Signup and view all the answers

    What is the end result of the PCR process?

    <p>Thousands to millions of copies of the DNA sequence</p> Signup and view all the answers

    What is the name of the device used to heat and cool the reaction tubes in PCR?

    <p>Thermal cycler</p> Signup and view all the answers

    Study Notes

    The Polymerase Chain Reaction (PCR)

    • A scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.

    Requirements of PCR Technology

    • The thermal cycler: heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction.
    • Template DNA: carries the DNA segment or target you wish to amplify.
    • Free nuclease water: must be present for the reaction to work.
    • Forward and Reverse Primers: short, single-stranded pieces of DNA that anneal to their complementary sequence on the template, providing initiation sites for DNA synthesis.
    • Thermostable DNA polymerase (e.g., Taq polymerase): synthesizes new strands of DNA by adding nucleotides onto the end of an annealed primer.
    • dNTPs: the four nucleotides used by DNA polymerase to extend an annealed primer (building blocks).
    • Magnesium: required for DNA polymerase activity (co-factor).
    • PCR buffer: necessary to create optimal conditions for Taq DNA polymerase activity.

    PCR Procedure

    • A series of 25-40 repeated temperature changes (cycles), each cycle consisting of:
      • Denaturation: heating the reaction to 94-97°C for 20-30 seconds to melt DNA.
      • Annealing: cooling the reaction to 50-65°C for 15-60 seconds to allow primers to bind to the template.
      • Extension/Elongation: heating the reaction to 72-80°C for extension time (dependant on DNA polymerase and DNA fragment length) to synthesize new DNA strands.
      • Final Elongation (optional): a single step at 70-74°C for 5-15 minutes to ensure full extension of any remaining single-stranded DNA.

    Applications of PCR

    • Medicine: enables early diagnosis of malignant diseases.
    • Classification of organisms.
    • Mutation detection.
    • Detection of pathogens.
    • Gene therapy.
    • Forensic science: important for the identification of criminals.
    • Evolutionary studies: plays an important role in phylogenetic analysis.
    • Analysis of ancient DNA from fossils.
    • Mapping the human (and other species) genome.
    • Isolation of a particular gene of interest from a tissue sample.
    • Generation of probes: large amounts of probes can be synthesized by this technique.
    • Production of DNA for sequencing.
    • Diagnosis of monogenic diseases (single gene disorders).
    • Detection of microorganisms: especially of organisms and viruses that are difficult to culture or take a long time.
    • Detection of microbial genes responsible for some aspect of pathogenesis or antibiotic resistance.
    • Crucial forensic evidence may often be present in very small quantities.

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    Description

    This quiz covers the polymerase chain reaction (PCR) technique in molecular biology, which amplifies specific DNA sequences for further study.

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