ANALYSIS OF DNA III.

POLYMERASE CHAIN REACTION

(PCR)

MARIANNA PAP

Medical Biology Department

 DNA AMPLIFICATION
= copy number of a specific DNA-fragment is increased

In vivo In vitro

Cloning of DNA-fragments – DNA-cloning Polymerase chain reaction (PCR)

Cloning of total genomic DNA – Genomic library
POLYMERASE CHAIN REACTION (PCR) TEMPLATE
5’ 3’
DNA replication O
 C C
• DNA polymerase not able to initiate DNA synthesis P C O
C C
C C G C
• requires a short oligonucleotide sequence complementary to the C O P
C C 
O
template DNA O
 C C
PRIMER P C O
• contains a free OH group at it’s 3’ end C C U A
C C
C O P
o DNA oligonucleotide (in vitro) C C
O

O

o RNA oligonucleotide (in vivo)
C C
P C O
C C
• direction of newly synthesized strand 5’ → 3’ 3’,5’ phosphodiester C C C G
C O P
bond C C 
O
OH
   C C
P-P-P C O
C
C
DNA polymerase C C G C
C O P
C C 
O
OH
   C C
P-P-P C O
Replication: template- and primer-dependent A T C C
C C
C O P
C C 
O
3’ 5’
POLYMERASE CHAIN REACTION (PCR)
The in vitro reaction mixture has to contain the following components:

• DNA fragment (which contains the region what we would like to amplify)
• 2 primers (short oligonucleotides, complementary to the DNA templates)
• heat-stable DNA polymerase → isolated from Thermus Aquaticus
bacteria live in hot springs called Taq polymerase
• 4 types of deoxyribonucleoside triphosphate (dATP, dTTP, dCTP, dGTP)
• buffer (provides the appropriate pH and salt concentrations)
POLYMERASE CHAIN REACTION (PCR)
Steps of the reaction
region to be amplified

3’ 5’
template DNA 5’ 3’

1. denaturation (~95°C)

3’ 5’

5’ 3’

2. primer binding (~55°C)

3’ 5’
primers 5’ 3’ 3’ 5’
5’ 3’

3. DNA synthesis (~72°C)

3’ 5’
5’ 3’
3’ 5’
5’ 3’

1st cycle
POLYMERASE CHAIN REACTION (PCR)
Steps of the reaction
region to be amplified

3’ 5’
template DNA 5’ 3’ 1. denaturation
3’ 5’
1. denaturation (~95°C) 5’ 3’
3’ 5’
3’ 5’
5’ 3’
5’ 3’
2. primer binding
2. primer binding (~55°C) 3’ 5’
5’ 3’ 3’ 5’
5’ 3’
5’ 3’ 5’
3’
5’ 3’
primers 5’ 3’ 3’ 5’ 3’ 5’
5’ 3’ 5’ 3’
3. DNA synthesis
3. DNA synthesis (~72°C) 3’
5’
5’
3’
3’ 5’
5’ 3’
3’ 5’ 3’ 5’
5’ 3’ 5’ 3’
3’ 5’ 3’ 5’
5’ 3’ 5’ 3’

1st cycle 2nd cycle

POLYMERASE CHAIN REACTION (PCR) 1. denaturation
3’ 5’
5’ 3’
Steps of the reaction 3’ 5’
region to be amplified 5’ 3’
3’ 5’
5’ 3’
3’ 5’ 3’ 5’
template DNA 5’ 3’ 1. denaturation 3’
5’
3’ 5’ 2. primer binding
3’ 5’
1. denaturation (~95°C) 5’ 5’ 3’
3’ 3’ 5’
3’ 5’ 5’ 3’
3’ 5’ 3’ 5’
5’ 3’ 5’ 3’
3’ 5’
5’ 3’ 5’ 3’
3’ 5’
2. primer binding 5’ 3’ 3’ 5’
2. primer binding (~55°C) 3’ 5’ 5’ 3’
5’ 3’ 3’ 5’ 3’ 5’
5’ 3’ 5’ 3’
3’ 5’
5’ 3’ 5’
3’ 5’ 3’
5’ 3’
primers 5’ 3’ 3’ 5’ 3’ 5’ 3. DNA synthesis
5’ 3’ 5’ 3’
3’ 5’
5’ 3’
3. DNA synthesis 3’ 5’
5’ 3’
3. DNA synthesis (~72°C) 3’
5’
5’
3’ 3’ 5’
3’ 5’ 5’ 3’
5’ 3’ 3’ 5’
5’ 3’
3’ 5’ 3’ 5’
5’ 3’ 3’ 5’
5’ 3’ 3’
3’ 3’ 5’ 5’
5’ 3’ 5’
5’ 3’ 5’ 3’ 5’ 3’
3’ 5’
5’ 3’
1st cycle 2nd cycle 3’ 5’
5’ 3’
At the end of cycle 1 the copy#: 2, at the end of cycle 2, copy#: 4, at the end of cycle 3, copy#: 8, at the end
of cycle 4, copy#: 16. After 30-40 cycles, billions more. 3rd cycle
POLYMERASE CHAIN REACTION (PCR)
PCR machine – thermocycler → computer-controlled incubator
Invented by K. Mullis (1983) – Nobel-prize in chemistry (1993)
Types of PCR
1. Traditional/conventional or end-point PCR
• amplified DNA fragments (amplicon) can be detected by agarose gel electrophoresis
• gel contains a DNA-binding fluorescence dye (e.g. ethidium bromide) → result can
be visualized under UV light
• the size and the amount of the amplicon can be detected
• disadvantage of the reaction: 1. 2.
-
o detection is possible only after the reaction is completed
1. marker
o the exact copy number of the amplified DNA fragment cannot be determined
2. amplicon

+
POLYMERASE CHAIN REACTION (PCR)
Types of PCR
1. Traditional/conventional or end-point PCR
2. Quantitative or real-time PCR (qPCR)
• Advantage: the amount of the amplified DNA fragments can be detected during the PCR reaction
• a fluorescent dye molecule is excited during the elongation step of every PCR cycle and the emitted signal is detected
by a detector
• cycle by cycle the amounts of fluorescence increase, which is proportional to the amount of the amplified DNA
fragments
• requires a specific PCR machine – able to able to excite the fluorescent dye and detect the emitted fluorescence
• 4-steps PCR: denaturation – primer binding – DNA-synthesis – fluorescence detection
POLYMERASE CHAIN REACTION (PCR)
2. Quantitative or real-time PCR (qPCR)
Detection of amplification:
a. double-stranded DNA binding fluorescent dye (SYBR green dye-based)

• reaction mixture contains all the components used in

region to be amplified
the traditional PCR
5’
template DNA 3’
5’ 3’
• + SYBR green dye - binds only to the double-stranded
1. denaturation
DNA 3’ 5’

• as double-stranded DNA fragments are synthesized 5’ 3’

during the reaction the amount of fluorescence 2. primer binding

3’ 5’
increases proportionally 5’ 3’
3’ 5’
SYBR green 5’ 3’
• advantage: cheap, easy to use → popular technique
3. DNA synthesis
• disadvantage: stains all double-stranded DNA in the 3’ 5’

reaction mixture, not only the amplicon excitation 5’ 3’ 3’ 5’

5’ 3’

4. fluorescence
emission detection
POLYMERASE CHAIN REACTION (PCR)
2. Quantitative or real-time PCR (qPCR)
Detection of amplification:
b. sequence-specific fluorescent reporter probe (TaqMan assay)
• name: "Taq" - taq polymerase, "Man" - PacMan game
region to be amplified
3’ 5’
• the reaction mixture contains all the components used in template DNA 5’ 3’

1. denaturation
the traditional PCR
3’ 5’
• + a probe complementary to one template strand 5’ 3’
2. primer and
o a fluorescent dye molecule (fluorophore) is bound fluorophore quencher
3’ 5’
probe binding
to it’s 5’ end 5’ 3’
3’ 5’
probe 5’ 3’
o a quencher molecule is bound to it’s 3’ end
o the quencher molecule inhibits the fluorescence of
the dye, if they are in close proximity
POLYMERASE CHAIN REACTION (PCR)
2. Quantitative or real-time PCR (qPCR)
Detection of amplification:
b. sequence-specific fluorescent reporter probe (TaqMan assay)
• Activity of DNA-polymerase:
region to be amplified
o 5’ → 3’ elongation activity
3’ 5’
o 5’ → 3’ exonuclease activity template DNA 5’ 3’

• the fluorophore is released → the amount of fluorescence can be 1. denaturation

detected 3’ 5’

5’ 3’
• the amount of fluorescence is proportional to the amount of the 2. primer and
amplified DNA fragments fluorophore quencher probe binding
3’ 5’
5’ 3’
3’ 5’
• the exact copy number of the amplified product can be determined probe 5’ 3’
at the end of each cycle
3. DNA synthesis
• advantage: specific for amplicon, multiple DNA sequences can be 3’ 5’

amplified in one reaction mixture (using multiple fluorophores) → 5’ 3’

multiplex PCR
excitation emission 4. fluorescence
• disadvantage: expensive
3’ 5’ detection
5’ 3’
POLYMERASE CHAIN REACTION (PCR)
2. Quantitative or real-time PCR (qPCR)
Detection of amplification:
b. sequence-specific fluorescent reporter probe (TaqMan assay)

qPCR amplification curve

• background baseline
• threshold or Ct: the point where the
fluorescence degree is significantly above the
background value
• exponential phase
• plateau phase (primer, dNTP runs out)
POLYMERASE CHAIN REACTION (PCR)
2. Quantitative or real-time PCR (qPCR)
Detection of amplification:
b. sequence-specific fluorescent reporter probe (TaqMan assay)

• suitable to compare the copy number of specific DNA sequences in different samples
• example: isolation of DNA from normal and tumor cells → qPCR amplification is performed using primers
and a probe specific for an oncogene sequence

• five-cycle difference → the amount of DNA

tumor cell DNA doubles in each cycle → the difference is 25 = 32
fluorescence

• the copy number of the specific oncogene

normal cell DNA
sequence in the tumor DNA sample is 32 times
treshold level higher, than in the normal DNA sample
0 5 10 15 20 25 30 35 40 45
Cycle number
POLYMERASE CHAIN REACTION (PCR)
Types of PCR:
1. Traditional/conventional or end-point PCR
5’ m7G A-A-A-A 3’ mRNA
2. Quantitative or real-time PCR (qPCR)
3. Reverse transcription PCR (RT-PCR) + reverse transcriptase
reverse transcription
• isolation of mRNA molecules + 4 dNTP (dATP, dTTP, dCTP, dGTP)
+ buffer
• synthesis of a complementary DNA (cDNA)
5’ m7G A-A-A-A 3’ mRNA
by reverse transcriptase 3’ T-T-T-T 5’ cDNA

• double-stranded cDNA molecules are used as mRNA degradation

templates in a traditional PCR reaction 3’ T-T-T-T 5’ cDNA

• agarose gel electrophoresis is used to detect 2nd strand synthesis
the amplified product 5’ A-A-A-A 3’
3’ T-T-T-T 5’
double-stranded cDNA
POLYMERASE CHAIN REACTION (PCR)
Types of PCR:
1. Traditional/conventional or end-point PCR
2. Quantitative or real-time PCR (qPCR)
3. Reverse transcription PCR (RT-PCR)
4. Reverse transcription quantitative PCR (RT-qPCR)
• isolation of mRNA molecules
• synthesis of a complementary DNA (cDNA) by reverse transcriptase
• double-stranded cDNA molecules are used as templates in a quantitative or real-time PCR reaction
• the amount of fluorescence is proportional to the amount of the amplified DNA fragments synthesized in the
reaction
POLYMERASE CHAIN REACTION (PCR)
Types of PCR:
1. Traditional/conventional or end-point PCR
2. Quantitative or real-time PCR (qPCR)
3. Reverse transcription PCR (RT-PCR)
4. Reverse transcription quantitative PCR (RT-qPCR)
5. Multiplex PCR
• multiple primer pairs specific to the DNA region are used in the same PCR reaction
• diagnosis of lesions affecting a larger DNA region (e.g. deletion, amplification)
• can be used in a conventional/endpoint PCR reaction or in a quantitative/real-time PCR reaction (qPCR)

Duchenne muscular dystrophy (DMD) B

Normal DMD
A -
Normal DMD gene E48
E44
E51
E44 E45 E46 E47 E48 E49 E50 E51
E45
DNA E50
E47
+
POLYMERASE CHAIN REACTION (PCR)
Application of PCR

• Probes for hybridization, DNA-fragments for sequencing

• DNA fingerprinting
o detects lots of microsatellite regions in the genome
o repeat number of those sequences is unique to an individual → DNA fingerprint
o used for DNA analysis in forensic sciences (identification of a person) or in paternity tests
• Diagnosis of inherited diseases or cancers – detection of point mutations, deletions, chromosomal translocation, gene
amplification
• Gene expression studies
• Diagnosis of infectious diseases – detection of the foreign nucleic acids
o Detection of DNA or RNA viruses e.g. HIV, influenza, HPV, Covid-19
o Detection of genomic DNA of bacteria e.g. tuberculosis
POLYMERASE CHAIN REACTION (PCR)
SARS-CoV-2 plate RT-qPCR
45+/94 specimens (48% positivity!)
October 24, 2020

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