6.3.5 Polymerase Chain Reaction Flashcards
1
Q
What actually is PCR?
A
- Where desired DNA/RNA is replicated
- In a test tube
2
Q
What are the requirements for PCR?
A
- Target DNA or RNA
- Primers (Tells DNA polymerase where to start building new strands, they are complementary to the 3’ end of DNA or RNA)
- DNA Polymerase (Taq polymerase)
- Free Nucleotides (to construct new strands of DNA or RNA)
- Buffer solution (For optimum pH)
- In a thermal cycler (provides optimal temperature a specific lengths of time for each stage of PCR)
3
Q
What are the key stages of PCR?
A
- Denaturation
- Annealing
- Elongation / Extension
4
Q
What is denaturation in PCR?
A
- First stage
- Double stranded DNA is heated to 95 degrees
- To break hydrogen bonds between existing single strands of DNA
5
Q
What is annealing in PCR?
A
- Second stage
- Temperature decreased to around 50-60 degrees
- Primers can anneal to 3’ ends of single DNA strands
6
Q
What is elongation / extension in PCR?
A
- Third stage
- Temperature at 72 degrees (for at least a minute)
- Optimal temperature for DNA Polymerase to build complementary strands of DNA
- Producing new identical double stranded DNA molecules
7
Q
Why is Taq polymerase used?
A
- Comes from a thermophilic bacterium
- So can withstand high temperatures
- So does not denature at high temps in Denaturation stage of PCR
- Optimal temp is high enough to prevent annealing of DNA strands that haven’t been copied yet
