Polymerase Chain Reaction

● Otherwise known as PRC, was developed by Kary Mullis to address the

disadvantages of cloning DNA using vectors and host cells (labor intensive and time
consuming)
● It is a rapid method of DNA cloning that extends the power of recombinant DNA and
in many cases eliminates the need to use host cells for cloning.
● It is a molecular biologic technique used to amplify a single copy of DNA to several
specific segments of DNA.
● A typical PCR reaction requires:
○ Template DNA
○ four deoxyribonucleoside triphosphates (dNTPs) which includes dATP, dGTP,
dTTP, dCTP
○ Taq Polymerase enzyme which is the thermal stable polymerase enzyme
○ Magnesium ion (Mg2+): an important cofactor for DNA polymerase
○ Forward Primer
○ Reverse Primer

The PRC Process

The amount of amplified DNA produced is theoretically limited only by the number of
times these cycles are repeated, although several factors prevent PCR reactions from
amplifying very long stretches of DNA. Most routine PCR applications involve a series of
three reaction steps in a cycle. These three steps are as follows:

1. Denaturation
● The double-stranded DNA to be cloned is denatured into single strands by
heating to 92 to 95°C for about 1 minute.

2. Hybridization/Annealing:
● The temperature of the reaction is lowered to between 45°C and 65°C,
which allows primer binding, also called hybridization or annealing, to the
denatured, single-stranded DNA.
 ● The primers serve as starting points for DNA polymerase to synthesize new
DNA strands complementary to the target DNA.
● When selecting a hybridization temperature for an experiment, factors such
as below are among primary considerations.
○ primer length
○ base composition of primers (GC-rich primers are more thermally
stable than AT-rich primers)
○ whether or not all bases in a primer are complementary to bases in
the target sequence

3. Extension:
● The reaction temperature is adjusted to between 65°C and 75°C, and DNA
polymerase uses the primers as a starting point to synthesize new DNA
strands by adding nucleotides to the ends of the primers in a 5′ to 3′ direction.

Each cycle results in amplification – a doubling of the number of DNA molecules

present at the start of the cycle. PCR is, therefore, a “chain reaction” because the products
of previous cycles serve as templates for each subsequent cycle.

Each PCR cycle takes 2 to 5 minutes and can be repeated immediately, so that in
less than 3 hours, 25 to 30 cycles result in over a million-fold increase in the amount of DNA.

PCR Machines
● Thermocycler is an instrument used to perform polymerase chain reactions.
● It can be programmed to carry out a predetermined number of cycles and automate
the process.