PCR - Amplification

Flashcards covering key concepts and details about the Polymerase Chain Reaction (PCR), including its components, process, and applications.

Who developed the Polymerase Chain Reaction (PCR)?

Kary Mullis, who received a Nobel Prize in 1993 for this technique.

What is the primary purpose of PCR?

To amplify a specific DNA segment through repeated cycles of synthesis.

What are the three main steps of PCR?

1. Denaturing, 2. Annealing, 3. Extension.

At what temperature does the denaturing step occur in PCR?

At 95°C.

What role do primers play in PCR?

Primers bind to the template DNA and initiate the synthesis of a new DNA strand.

What is Taq polymerase?

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

What is the significance of the temperature during the annealing step of PCR?

The temperature is ideally 2-4°C below the melting temperature (Tm) of the primer pair.

Describe the term 'processivity' in the context of DNA polymerases.

Processivity is an enzyme's ability to catalyze consecutive reactions without releasing its substrate.

What can PCR be used for in molecular diagnostics?

Genotyping, prenatal diagnosis, and pathogen detection.

What effect does the number of cycles have on DNA amplification in PCR?

Starting from the third cycle, the amount of double-stranded product increases exponentially by 2^n.

What are the requirements for DNA polymerase to synthesize DNA?

A pre-existing DNA or RNA strand (primer), a template DNA strand, incoming dNTPs, and Mg2+.

What is the purpose of adding mineral oil during PCR?

To overlay the sample if using a PCR machine without a heated lid, preventing evaporation.

What are some applications of PCR in archaeology?

Molecular identification and DNA fingerprinting.

What is considered an important characteristic of PCR primers?

They should be 18-24 bases long and have a melting temperature (Tm) of 50-60°C.

What can inhibit PCR reactions?

Materials such as SDS, EDTA, and other compounds present in template DNA.

Why is processivity important for DNA polymerase during PCR?

It affects the average number of nucleotides added per encounter with the template strand.