The Polymerase Chain Reaction

1
The Polymerase Chain Reaction
2
Kary Mullis invented a technique called the
polymerase chain reaction or PCR for short. PCR
makes billions of copies of DNA from an original
tiny sample. The CSI people would call this DNA
amplification.
3
Mullis received the Nobel Prize for his discovery
of the PCR technique in 1993.
4
Assignment 18 has a copy of this table. Complete
each row of the table as each cycle of the
polymerase chain reaction is completed.
5
Suppose this is the portion of DNA to be
amplified.
Cycle 1 The DNA is denatured by heating to 95oC
DNA primers are included with the DNA sample.
DNA primers bind to DNA when cooled to 60oC
Taq polymerase along with deoxynucleotides are
also part of the mixture.
5' A T G C T T G C A A T C C G A T T C A C C G C
T 3'
3' A A G T G 5'
5' G C T T G 3'
3' T A C G A A C G T T A G G C T A A G T G G C G
A 5'
6
Taq polymerase is named after the bacterium from
which it is obtained. Thermus aquaticus (also
Thermophilus aquaticus) is a chemosynthetic
bacterium that lives in hot springs. All enzymes
made by T. aquaticus are stable at high
temperatures.
Thermus aquaticus Bactéries déposées sur un
filtre millipore de 0,22 µm (échelle1 µm)
Photo Diane Montpetit (Centre de recherche et
de développement sur les aliments, Agriculture et
Agroalimentaire Canada)
7
Cycle 1
When the mixture is warmed to 72oC, Taq
polymerase produces the complementary DNA strand
in the 5 to 3 direction when.
5' A T G C T T G C A A T C C G A T T C A C C G C
T 3'
3T A C G A A C G T T A G G C T
A A G T G 5'
3A A G T G 5'
C A A T C C G A T T C A C C G C T3
5' G C T T G 3
5' G C T T G
3' T A C G A A C G T T A G G C T A A G T G G C G
A 5'
8
DNA is heated to 95oC again and denatured.
Cycle 2
Primers are annealled to DNA strands when cooled
to 60oC.
Taq polymerase extends the strands when warmed to
72oC.
3 T A C G A A C G T T A G G C T
C A A T C C G A T T C A C 3
5' A T G C T T G C A A T C C G A T T C A C C G C
T 3'
3T A C G A A C G T T A G G C T
A A G T G 5'
5' G C T T G
C A A T C C G A T T C A C C G C T3
3' T A C G A A C G T T A G G C T A A G T G G C G
A 5'
3 C G A A C G T T A G G C T
C A A T C C G A T T C A C C G C T 3
Notice the appearance of strands of DNA of
different length. These are referred to as
variable length fragments.
5' G C T T G
A A G T G 5'
5' G C T T G
A A G T G 5'
9
Repeat the cycle again. That is Heat to 90oC
to denature the DNA Cool to 60oC to anneal the
primers Warm to 70oC to allow Taq polymerase to
extend the primers. and copy the DNA through 30
cycles.
Repeating the cycle 30 times yields exponential
growth in the number of copies of DNA as given by
the relation Number of copies of DNA
2number of cycles
230 1 073 741 824 Of this total,
1 073 741 764 are copies of the short target
sequence of DNA.
10
The Thermal Cycler The repetitive nature of
heating and cooling lends itself to automation.
Table top thermal cyclers are no larger than a
bread-baking machine. The thermal cycler will
take a mixture consisting of the original DNA
sample, Taq polymerase, forward and reverse
primers and free deoxynucleotides, all in a 0.5
mL tube and produce a billion copies in just a
few hours. The thermal cycler illustrated here
can hold 25 tubes. The white caps of four tubes
are visible in this photo.
11

• Note the display
• the thermal cycler is programmed for 20 cycles
• DNA is denatured at 94oC for 30 s
• primers anneal at 55oC for 1 minute
• and Taq polymerase is given 3 minutes at 68oC to
  extend the primers and produce the complementary
  strand.

12
PCR Animations
http//www.dnalc.org/ddnalc/resources/animations.h
tml
http//www.amnh.org/learn/pd/genetics/pcr/
Textbook References Nelson Biology 12 6.3
Advanced Molecular Biological Techniques, pp 296
298 Campbell Biology, pp 382 - 383