PCR – fast and inexpensive DNA amplification; molecular photocopying
Series of 20-40 repeated cycles of DNA replication by a DNA polymerase enzyme
Single DNA strand, over 1 trillion copies are generated from a 40 cycle reaction
Process
1.
Initialization a.
Reaction heated to 94-96 C for 2-10 mins (Activates the DNA polymerase and denatures contaminants)

2.
Denaturation a.
Hydrogen bonds between the double stranded DNA are broken by heating to 94 to 98
C for 20 to 30 sec
3.
Annealing a.
Primers bind to a complementary sequence in the DNA template to guide DNA polymerase replication at 50 to 65C for 20 to 40 seconds
4.
Elongation a.
DNA polymerase starts to incorporate dNTPs in a 5’ to 3’ direction to synthesize a new
DNA strand. The temperature and extension time depends on the type of DNA polymerase enzyme and the target amplicon. i.
Taq polymerase (1min/1-1.5kilobases; 72-78C)
5.
The cycle then repeats from denaturation to elongation 20-40 times. At the end of the last cycle, there is a final elongation step that keeps the reaction mixture at 72-78C for 5-15 mins.
This ensures that any remaining DNA strands are fully extended. A final holding step keeps the mix at 4-15C for an indefinite time, keeping thr products for short term storage.