POLYMERASE CHAIN REACTION (PCR)
Goal of PCR: to make a massive number of identical copies of a targeted DNA sequence
Involves 3 Different Steps – repeated in a series of many cycles
1. Denaturing the DNA
2. Annealing the primers
3. Primer extension
At completion of each cycle, the number of target DNA molecules is doubled
PCR reaction does not amplify all DNA – amplifies a specific target sequence of interest
First Step: Denaturation of DNA
The double helix must be separated into single strands
Heating the DNA to about 92-95 degrees C
Denatures or breaks the hydrogen bonds that hold strands together
Second Step: Anneal primers
Quickly lower temperature to about 45-65 degrees C
Lower temperature allows for a pair of primers to anneal or hybridized to the target DNA
via complementary base pairing
Primers: single-stranded molecules of DNA, often 20 nucleotides long, with a specific
sequence that is to be complementary to the ends of the target sequence
Starts at the 5’ end of the primer on one strand, and ends at the 5’ end of the primer on the
other stand
5’ ends of the primers define the ends of the target sequence that is being amplified
Third Step: Primer extension
Raise temperature to about 65-75 degrees C
Ideal temp for optimal DNA polymerase function
DNA polymerase synthesizes new strands of DNA by extending off the ends of each
primer in a 5’ to 3’ direction
New synthesized strands extend beyond the target area in the first cycle
Process can be repeated for exponential amplification
First cycle produces two DNA molecules for every one DNA molecule that is present at
the start of the reaction – doubling
Second cycle, number of molecules are doubled again – now four molecules of the target
for each original molecule of the template DNA
Third cycle doubles the DNA products again – produces 8 molecules of the target for
each original molecule of the template DNA
Many products of cycle three have strands that extend beyond target region
