Polymerase Chain Reaction (PCR) in Molecular Diagnostic Microbiology
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Questions and Answers

PCR is a laboratory technique for producing millions to billions of copies of RNA.

False (B)

PCR is also known as molecular photocopying.

True (A)

PCR is a slow and expensive technique.

False (B)

The main activity of DNA polymerase is to cut DNA strands.

<p>False (B)</p> Signup and view all the answers

Primers bind to the template RNA during PCR.

<p>False (B)</p> Signup and view all the answers

PCR can be used to produce billions of copies of a specific segment of DNA for detailed study.

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

Pfu polymerase is derived from Pyrococcus furiosus.

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

Pfu polymerase has 5' to 3' exonuclease activity.

<p>False (B)</p> Signup and view all the answers

Pfu polymerase is characterized by high processivity compared to Taq polymerase.

<p>False (B)</p> Signup and view all the answers

1–2 units of Taq DNA polymerase are sufficient for a typical 50 µL PCR reaction.

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

Increasing the amount of DNA polymerase in a reaction with inhibitors may reduce PCR yields.

<p>False (B)</p> Signup and view all the answers

Taq Polymerase has terminal transferase activity at room temperature.

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

Magnesium ions function as a cofactor for the activity of RNA polymerases.

<p>False (B)</p> Signup and view all the answers

Mg2+ facilitates the formation of complex between primers and DNA templates by destabilizing negative charges on their phosphate backbones.

<p>False (B)</p> Signup and view all the answers

Potassium ions from KCl promote primer annealing in PCR.

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

Ammonium sulfate can replace KCl in the buffer to enhance specificity in PCR.

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

Denaturation phase in PCR involves cooling the dsDNA template at 94-95 °C.

<p>False (B)</p> Signup and view all the answers

Low Mg2+ concentrations in PCR result in enhanced stability of primer-template complexes.

<p>False (B)</p> Signup and view all the answers

Multiplex PCR involves amplifying multiple targets using a single primer pair.

<p>False (B)</p> Signup and view all the answers

Distinct amplicons in conventional PCR need to have similar base pair lengths for visualization.

<p>False (B)</p> Signup and view all the answers

Nested PCR increases the specificity of DNA amplification by reducing non-specific amplification of DNA.

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

A nested PCR assay typically has three sets of primers for a single locus.

<p>False (B)</p> Signup and view all the answers

The first PCR run in nested PCR uses the inner pair of primers.

<p>False (B)</p> Signup and view all the answers

Nested PCR results in the production of a longer PCR product compared to regular PCR.

<p>False (B)</p> Signup and view all the answers

During the extension step in PCR, DNA polymerase extends the primer sequences from the 5’ end of each primer.

<p>False (B)</p> Signup and view all the answers

A 2 min extension is typically sufficient to synthesize PCR fragments up to 3 kilobases (kb).

<p>False (B)</p> Signup and view all the answers

During the first extension in PCR, the template will be length limiting, restricting the synthesis of templates exceeding the amplicon length.

<p>False (B)</p> Signup and view all the answers

A typical PCR reaction contains 20–30 cycles in a standard PCR run and can increase up to a maximum of 50 cycles.

<p>False (B)</p> Signup and view all the answers

Increasing the number of cycles in PCR with large amounts of starting material is advisable to ensure higher yield of PCR product.

<p>False (B)</p> Signup and view all the answers

At the end of a PCR reaction, amplification products are typically analyzed using mass spectrometry.

<p>False (B)</p> Signup and view all the answers

Flashcards

PCR amplifies RNA

PCR (Polymerase Chain Reaction) is a technique used to amplify specific DNA sequences, not RNA.

PCR is slow and expensive

PCR is a powerful and versatile technique for amplifying DNA, making it a fundamental tool in molecular biology research.

DNA polymerase cuts DNA strands

DNA polymerase is an enzyme that adds nucleotides to a growing DNA strand, not cut them.

Primers bind to template RNA during PCR

During PCR, primers bind to complementary sequences on the DNA template, not RNA.

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PCR produces copies of RNA

PCR produces billions of copies of a specific segment of DNA, not RNA.

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Pfu polymerase origin

Pfu polymerase is an enzyme extracted from the archaeon Pyrococcus furiosus.

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Pfu polymerase has 5' to 3' exonuclease activity

Pfu polymerase does not possess the 5' to 3' exonuclease activity, unlike Taq polymerase.

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Pfu polymerase has high processivity

Taq polymerase is known for its high processivity, whereas Pfu polymerase is characterized by higher fidelity.

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Taq polymerase units in a reaction

1–2 units of Taq DNA polymerase is sufficient for a typical 50 µL PCR reaction.

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Increasing DNA polymerase reduces PCR yield

Increasing the amount of DNA polymerase in a reaction with inhibitors can compensate for the inhibition, ensuring optimal PCR yields.

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Taq polymerase has terminal transferase activity

Taq polymerase displays terminal transferase activity at room temperature, adding nucleotides to the end of a DNA strand without a template.

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Mg2+ cofactor for RNA polymerase

Magnesium ions are cofactors for DNA polymerase, not RNA polymerase.

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Mg2+ destabilizes phosphate backbones

Mg2+ facilitates DNA polymerase activity by stabilizing the negative charges on the DNA backbone, enabling primer binding.

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Potassium ions promote primer annealing

Potassium ions from KCl help in primer annealing by promoting their interaction with DNA template.

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Ammonium sulfate replaces KCl in the buffer to enhance specificity in PCR

Ammonium sulfate can improve specificity in PCR by replacing KCl in the buffer, reducing non-specific primer binding.

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Denaturation phase involves cooling dsDNA

The denaturation phase involves heating the dsDNA template at 94-95 °C to separate the strands, not cooling.

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Low Mg2+ concentration results in increased stability of primer-template complexes

Low Mg2+ concentrations in PCR lead to decreased primer-template complex stability, making it harder for primers to bind.

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Multiplex PCR uses a single primer pair

Multiplex PCR amplifies multiple targets simultaneously using multiple primer pairs, not a single pair.

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Distinct amplicons need similar base lengths to visualize

Distinct amplicons in conventional PCR can have different base pair lengths for visualization.

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Nested PCR increases specificity by reducing non-specific amplification of DNA

Nested PCR increases specificity by using two sets of primers, the outer primers amplifying a larger region, and the inner primers amplifying a smaller, nested region.

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Nested PCR uses three primer sets for one locus

A nested PCR assay typically uses two sets of primers: an outer set for the first PCR and an inner set for the second PCR.

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The first PCR run uses inner pair of primers

The first PCR run in nested PCR uses the outer pair of primers, amplifying a larger region.

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Nested PCR produces a longer PCR product

Nested PCR results in a shorter PCR product compared to regular PCR, as the inner primers amplify a smaller region within the outer amplified region.

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DNA polymerase extends primer sequences from the 5' end

During the extension step, DNA polymerase extends the primer sequences from the 3' end of each primer, adding nucleotides to the growing DNA strand.

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A 2 min extension is sufficient for 3kb fragments

A minimum of 3 minutes is typically required for synthesizing PCR fragments up to 3 kilobases, depending on the polymerase used and PCR conditions.

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The template is not length limiting in the first extension

During the first extension, the template concentration limits the amount of product exceeding the amplicon length.

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A typical PCR run contains 20-30 cycles and can go up to 50 cycles

A standard PCR run typically contains 20–30 cycles, with a maximum of 40 cycles commonly used in most PCR reactions.

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Increasing the number of cycles is advisable when starting material is high

Increasing the number of cycles in PCR with large amounts of starting material can lead to saturation of products, compromising the yield.

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PCR products are analyzed using mass spectrometry

Electrophoresis, a process that separates molecules based on size and charge, is commonly used to analyze PCR products, not mass spectrometry.

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