lecture 2 PCR Fundamentals and Quantification

These flashcards cover key concepts from the lecture on PCR fundamentals, quantification techniques, and best practices to prevent contamination.

What is PCR instrumental to?

Quantitation process.

What does PCR stand for?

Polymerase Chain Reaction.

Who won the Nobel Prize for discovering PCR?

Kary Mullis.

In PCR, what does the 'N' in DNTPs stand for?

Adenine (A), Guanine (G), Cytosine (C), or Thymine (T).

What is a crucial step in the PCR set-up process?

Including a positive control, a negative control, and an extraction control.

At what temperature does the initial denaturation step of PCR take place?

95 degrees Celsius.

What does real-time PCR measure?

The amount of DNA generated during the PCR process.

What does CT value represent in real-time PCR?

The number of cycles it takes for fluorescence to reach a threshold.

Why is quantification of DNA important in PCR?

To ensure the correct amount of DNA is used for accurate analysis.

What type of contamination is most dangerous in PCR protocols?

PCR product contamination.

What is the function of magnesium ions in PCR?

They assist in the enzymatic activity of polymerase.

What's the role of primers in PCR?

They bind to specific sequences of DNA to initiate amplification.

What happens during the extension phase of PCR?

Polymerase extends the new DNA strand from the primer.

How does PCR achieve exponential amplification?

Each cycle doubles the amount of target DNA.

What is multiplexing in PCR?

The ability to amplify multiple DNA regions in a single PCR reaction.

What is a common way to assess DNA quality before PCR?

Using spectrophotometry to measure absorbance.

What influences the melting temperature of DNA primers?

The GC content and length of the primers.

What can cause amplification failure in PCR?

Degraded DNA samples or presence of inhibitors.

What are the consequences of using too much DNA in PCR?

It may create too much noise in analysis, complicating interpretation.

What is one way to minimize contamination in PCR?

Having separate rooms for extraction and PCR setup.