Polymerase Chain Reaction

PCR
DNA structure
 History of PCR
Kary Mullis
Envisioned the technique in 1983: He thought up this
protocol to save a project he was about to lose because
he had used up virtually all his sample DNA
Cetus offered him a $10,000 bonus
The method was first described at a conference in 1985
US Patent #s 4,683,202; 4,683,195; 4,965,188: Process for
amplifying, detecting, and/or cloning nucleic acid
sequences using a thermostable enzyme 1987

Discovery of Taq
Thermus aquaticus discovered in 1967
Taq named molecule of the year (Science) in 1989
Withstand a temperature of 95oC - extremozyme
 What is Polymerase Chain Reaction
The amplification of a DNA region through thermocycling
using a heat stable DNA-polymerase and synthetic primers
A synthesis method to specifically amplify regions of which the
respective sequence ends are known
Procedure
template DNA
a big surplus of 2 synthetic oligonucleotides (primer),
complementary to the two ends of the sequence to be
amplified
temperature resistant DNA-polymerase (Taq-Polymerase
of Thermus aquaticus)
a big surplus of desoxyribonucleotides (dNTPs)
Temperature cycles
denaturing
annealing
elongation
Polymerase Chain Reaction: Principle of the Method
Denaturation
Elongation
Annealing
5`
I. 3`

3` 5`

II.
5` 3`

3` 5`

III.
5` 3`

3` 5`
Not amplified at all

Linear amplification: 2x no. of cycles

Exponential amplification: 2no. of cycles

Polymerase Chain Reaction: Principle of the Method
Newly synthesized DNA-strands also serve as template and thus
contribute to an exponential amplification (2n)
 Polymerase Chain Reaction: Kinetics

I II III I: early phase: initial

product concentration

binding of primers and

product formation

II: mid phase: optimal

amplification and
exponential product
formation

III: Late or plateau

phase: suboptimal
amplification
PCR - cycles
 Polymerase Chain Reaction: Parameters
What do we need:

DNA as template

Taq-DNA Polymerase as the synthesis tool

Primer as starting points for DNA-synthesis

dNTPs (dATP, dTTP, dGTP, dCTP) as

building blocks

Buffer

Thermocycler
Polymerase Chain Reaction: Parameters
Reaction volume: 100 L
Taq Polymerase: 2-2.5 U
Primer: 0.1-1.0 M
Salt: 6-50 mM KCl
Mg++: 1.5-5.0 mM MgCl2 or
MgSO4
Buffer: Tris-HCl 10-50 mM, pH
7.5-9.0
Template DNA 102-106 copies
 Polymerase Chain Reaction: Primer

Taq requires a 3OH for the start of elongation

The GC-content dictates the annealing temperature

Secondary structures (hair-pin loops) should be avoided

 Polymerase Chain Reaction:
Thermocycling
Typical Thermocycling Protocol
Denature DNA-template at 95C
Anneal between 50C and 65C to allow
hybridization of the primers to their specific
complementary sequences
Elongation at 72C to enable polymer elongation
repeat 20 to 30 times
Last Elongation step to complete amplicon
synthesis
 Polymerase Chain Reaction: Optimization

Optimal concentration of Mg 2+
, other ions, dNTPs and
polymerase
Efficient denaturation
High annealing temperature
Use of additives (DMSO, formamide, etc.)
Elimination of PCR inhibitors
"Nested" PCR
Special PCRs: "Touchdown", "Hot-Start
Other things to consider:
PCR tubes
Thermocycler
 Polymerase Chain Reaction: Polymerases

Taq-Polymerase: isolated from Thermus aquaticus -

The classical polymerase

Tth-Polymerase: isolated from Thermus thermophilus

with high reverse transcription activity
Pfu-, Pwo-Polymerase: isolated from Pyrococcus
furiosus or P. woesei proofreading activity
Different "Hot-Start" Taq-Polymerases
modified Taq-Polymerases, activation after heating
to 95C
PCR prevention of contamination

Contamination by
- Template DNA

- Cloned DNA fragments

- Amplified DNA fragments

PCR prevention of contamination
Spatial separation of the working steps
(DNA extraction, preparation of reaction
mix, PCR, analysis of PCR products)

Use of pipettes only within these

compartments

Change of gloves between compartments

Workbench with UV-light for preparation
of PCR reaction mix
PCR prevention of contamination

Only filter tips

Only autoclaved reagents
All reagents in aliquots and use only
once

Regular cleaning of all devices and

work places

Controls: positive, negative and

extraction
 Polymerase Chain Reaction: Significance of the Method
Applications
1. Clinical diagnostics
a. Detection of mutations
prenatal diagnostics
hereditary diseases
cancer
b. Infectious diseases
pathogen detection
pathogen search
2. Forensics
3. Evolutionary relationships
4. Tool in molecular biology
direct sequencing
site-directed mutagenesis
gene technology (cloning,
fusion proteins, etc.)
5. Economic importance

Publication Nobel Prize

1987 Chemistry
1993