L12 PCR Flashcards
(59 cards)
What does PCR stand for?
polymerase chain reaction
What is the purpose of PCR?
to amplify a specific DNA fragment
What enzyme is essential for PCR?
DNA polymerase (e.g. Taq polymerase)
What are the essential components of a PCR reaction?
Template DNA, primers, dNTPs, buffer with Mg²⁺, DNA polymerase
What does dNTP stand for?
deoxyribonucleoside triphosphate
What is the role of primers in PCR?
They provide a starting point for DNA synthesis
What is the typical length of PCR primers?
18-25 nucleotides
Why are primers added in excess in PCR?
To favour primer-template binding over strand re-annealing
What do universal primers bind to?
abundant sequences
What do specific primers bind to?
a target DNA fragment
What is the function of Mg2+ in PCR buffer?
primarily functions as a cofactor for DNA polymerase, facilitating the incorporation of dNTPs
Why is Taq polymerase used in PCR?
it is thermostable and functions at high temperatures
What equipment is used to control PCR temperatures?
a thermal cycler
What is the first step in PCR?
initialisation at 94°C to break secondary structures
What happens during PCR denaturation
DNA strands are separated at ~94°C
What is the annealing step in PCR?
primers bind to single-stranded DNA
What temperature is annealing typically performed at?
30-60°C, depending on primers
What occurs during extension in PCR?
DNA polymerise synthesises new DNA at 68-72°C
What is the purpose of the final elongation step?
To complete any remaining DNA synthesis
What is the typical hold temperature after PCR?
4°C
When are the first short, desired-length PCR products formed?
In the third cycle
How many DNA copied are produced after 36 PCR cycles?
2^36 or ~68 billion
Why does PCR amplification plateau?
due to reagent depletion
What can be done if more DNA copies are needed after 35 cycles?
Re-amplify the product in another PCR round
