Chapter 4 : Polymerase Chain Reaction (PCR)

Rekayasa Biomolekuler

Mochamad Untung Kurnia Agung, S.Kel., M.Si.

Marine Molecular Biotechnologist

Polymerase Chain Reaction (PCR)

PCR
PCR is a laboratory
version of DNA
Replication in cells

History of PCR
PCR was invented in 1983 by Dr. Kary Mullis,
for which he received the Nobel Prize in
Chemistry in 1993.

Mullis, born in Lenoir, North

Carolina, attended the University of
Georgia Tech for his undergraduate
work in chemistry, and then
obtained a Ph. D. in biochemistry
from Cal Berkeley.

Mulliss Nobel Prize, 1993

Initially PCR used the Klenow fragment of E.coli

DNA polymerase inactivated by high
temperatures
Kleppe, Ohtsuka, Kleppe, Molineux, Khorana. 1971. J. Mol. Biol.
56 : 341

95 C
5 min

55 C
3 min

35 times

8 BORING hours per PCR!

72 C
5 min

Thermocyclers

heated lids
adjustable ramping times
single/multiple blocks
gradient thermocycler blocks

Components of PCR Reaction

Target DNA

contains the sequence to be amplified

Pair of Primers

oligonucleotides that define the sequence

to be amplified

dNTPs

deoxynucleotidetriphosphates: DNA building

blocks

DNA Polymerase
Mg++ ions
Buffer Solution

enzyme that catalyzes the reaction

cofactor of the enzyme
maintains pH and ionic strength of the reaction
solution suitable for the activity of the enzyme

Steps of PCR Reaction

The basis of PCR reaction is temperature changes and
the effect that these temperature changes have on the
DNA
Step 1

Denature DNA

At 94 - 95 C,
the DNA is denatured (i.e. the two strands are
separated)

Step 2

Primers Anneal

At 40C- 65C,
the primers anneal (or bind to) their
complementary sequences on the single
strands of DNA

Step 3

DNA Polymerase At 72C,

extends the DNA DNA Polymerase extends the DNA chain by
chain
adding nucleotides to the 3 ends of the
primers

Typical
Thermal
Cycler
Conditions

1.
2.
3.
4.
5.
6.

Initial Denaturation
95 C
DNA Denaturation
95 C
Primer Annealing
65 C
Primer Extension
72 C
Go to step #2, repeat 39 more
End

3 min
1 min
1 min
1 min
times

PCR Primers
Primer is an oligonucleotide sequence will
target a specific sequence of opposite base
pairing (A-T, G-C only) of single-stranded
nucleic acids

Reverse Primer
Forward Primer

Primers Specifity
Universal

amplifies ALL organism DNA

for instance
Ex. 16S rRNA for ALL bacteria

Group Spesific

amplify ALL groups for instance

Ex. Nitrificier group of bacteria

Spesific

amplify just a given sequence

PCR Result

Gel Electrophoresis

Next :
Mid Term Test (UTS)