Polymerase Chain Reaction (PCR) in Molecular Diagnostic Microbiology

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

False

PCR is also known as molecular photocopying.

True

PCR is a slow and expensive technique.

False

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

False Signup and view all the answers

Primers bind to the template RNA during PCR.

False Signup and view all the answers

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

True Signup and view all the answers

Pfu polymerase is derived from Pyrococcus furiosus.

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Pfu polymerase has 5' to 3' exonuclease activity.

False Signup and view all the answers

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

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

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

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Taq Polymerase has terminal transferase activity at room temperature.

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

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

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Potassium ions from KCl promote primer annealing in PCR.

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

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

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

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

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

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

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

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

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

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

False Signup and view all the answers

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

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

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

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

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

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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.

PCR is also known as molecular photocopying.

PCR is a slow and expensive technique.

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

Primers bind to the template RNA during PCR.

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

Pfu polymerase is derived from Pyrococcus furiosus.

Pfu polymerase has 5' to 3' exonuclease activity.

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

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

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

Taq Polymerase has terminal transferase activity at room temperature.

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

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

Potassium ions from KCl promote primer annealing in PCR.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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