Principles of Molecular and Immunologic Studies - Nucleic Acid Amplification

This set of flashcards covers key concepts, processes, and components of nucleic acid amplification, specifically focusing on PCR, its principles, applications, controls, and potential variables impacting results.

What is the principle of polymerase chain reaction (PCR)?

A primer-directed in vitro enzymatic reaction for the production of a specific DNA fragment.

What are the three main steps of PCR?

Denaturation, annealing, and extension.

What is Taq polymerase?

A thermostable DNA polymerase used in PCR for synthesizing new DNA strands.

What is the significance of heat-stable enzymes in PCR?

They allow the reaction to withstand high temperatures needed for denaturation without losing activity.

What is evaluated when visualizing PCR products by gel electrophoresis?

To determine if the product is the desired amplicon or a result of primer dimers or mispriming.

How does the number of cycles affect PCR product yield?

The copy number theoretically doubles at the end of each cycle, leading to exponential amplification.

What conditions are required for successful PCR?

Specific annealing temperatures, appropriate enzyme concentration, and correct buffer conditions.

What is the importance of primer design in PCR?

Primers determine the specificity and efficiency of the reaction, as well as the size of the PCR product.

What are PCR controls and why are they important?

Controls that assess specificity, sensitivity, and contamination in PCR experiments.

What is the role of dNTPs in PCR?

Deoxynucleotide triphosphates serve as building blocks for the synthesis of new DNA strands.

What is the purpose of a reagent blank in PCR controls?

To control for contamination by containing all reagents except the DNA template.

Explain the concept of amplification in PCR.

PCR amplifies the copy number of a specific DNA sequence from a few initial copies to millions.

What does a melting curve indicate in PCR?

It indicates the temperatures at which different DNA strands denature, providing information on the specificity of the PCR products.

What are the potential sources of contamination in PCR?

Amplicons from previous reactions or unintended DNA sources that can affect results.

How do you ensure the specificity of PCR amplification?

By designing high-quality, specific primers and including proper controls.

What is the significance of the primer dimers formation?

They can complicate results by producing non-specific products that appear in gel electrophoresis.