Polymerase Chain Reaction Flashcards
What are the essential ingredients that must be present for PCR to occur? (3•)
•Taq Polymerase
•dNTPs
•Primers
How long are primers usually in PCR?
20 nucleotide bases.
Where does Taq polymerase come from?
Hot-spring bacteria, Thermus aquaticus.
How many cycles occur in PCR?
About 20-35 cycles.
Cycle 1?
•Cycle 1: 95c -> 72c
•Denatures DNA by breaking the hydrogen bonds that hold them together. •Then, it adds nucleotides from the 5’-3’ direction.
Cycle 2?
•Temperature: 72c
•Duplicates the duplicated strands again due to excess primers and nucleotides.
Cycle 3?
A total of 8 copies of target DNA while 2 copies are just the target sequences.
After each PCR cycle, what happens to the number of copies of our target sequences?
The number of copies of our target sequences doubles.
What is the main goal of PCR?
To create many copies of a piece of DNA.
Steps of amplification?
•Denaturation: separates double-stranded DNA by heating it.
•Annealing: Solution is cooled and the two primers go to their complementary sequence on the strands of the template duplex.
•Extension: DNA polymerase synthesizes new DNA strands in the 5’ to 3’ direction.
How many cycles are typical in PCR?
25-35 cycles.
What is species about cycle 4?
Half of the fragments consist of just target dna and the other half contains flagging dna
