301-8245 PCR Basics I Rev B

Basics of
Polymerase Chain Reaction

(PCR)
1 © 2017-2020 Cepheid - All rights reserved - 301-8245, Rev B, November 2020,

Agenda
Basics of Molecular Biology

PCR Basics Part I Definition of PCR
The phases of PCR
Definition of Real Time PCR
PCR Basics Part II
Qualitative Real time PCR
Quantitative Real Time PCR
PCR Basics Part III Definition of Melting Temperature

Melt Curve Analysis
Learning Objectives
The General Objective of this Module Is to Give You an

Understanding of the PCR methods used with the GeneXpert
At the end of the training, you will be able to:
• List the elements involved in the PCR Process

• Explain the PCR Process and describe the PCR steps
• Define “RT-PCR” (2 possible meanings)
• Describe the RT-PCR curves, define the Ct
• Explain how quantitation can be performed with RT-PCR
• Define Melting Temperature
• Explain how Melting Curve Analysis allows to identify microbial resistance

Basics of
Polymerase Chain Reaction
(PCR) - I

Basics of Molecular
Biology
A quick reminder
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2020
Basics of Molecular Biology
Genetic information contained in DNA
▪ DNA is built in a double helix

▪ DNA encodes the genetic
information (different genes,
different information)
▪ DNA is organized into a long chain
called chromosomes
▪ 23 pairs of chromosomes are in the
nucleus of human cells
Genetic material in bacteria
• Genetic information in bacteria is encoded in DNA

• Most bacteria have a genome that consists of a single circular DNA molecule,
located in a region called nucleoid (not bound by a membrane)
• Extrachromosomal genetic elements such as plasmids and bacteriophages often
determine resistance to antimicrobial agents, production of virulence factors, or
other functions.

Genetic material in viruses
• A virus is a small parasite that cannot reproduce by itself. It relies on the host
cell machinery.
• Viral genome can be found in various forms : RNA or DNA, single or double
stranded, linear circular or even segmented
Dreamstime
Ex: Hepatitis C virus
DNA building blocks
DNA is made up of 4 nucleotides

‒ A = Adenine
‒ T = Thymine
‒ C = Cytosine
G A T C
‒ G = Guanine
The 4 bases are linked together to form a sequence (single strand of DNA)
G C T T A T G C A T

Basic Molecular Biology
Most DNA is double stranded and pairs in a unique way:
G C T A
Antisense strand 3’ 5’
Hydrogen bonds
C G A A T A C G T A
G C T T A T G C A T
Sense strand
5’ 3’

DNA replication
• New DNA is made by enzymes called DNA

polymerases. They synthesize DNA in the 5'
to 3' direction only.
• Another enzyme called primase makes an
RNA primer to prime the Polymerase.
• Once the RNA primer is in place, DNA
polymerase "extends" it, adding nucleotides
one by one to make a new DNA strand that is
complementary to the template strand.

Definition of PCR
12 2020,
© 2017-2020 Cepheid - All rights reserved - 301-8245, Rev B, November 2020
What is PCR ?
1. PCR (Polymerase Chain Reaction) is a chain reaction which generates

multiple copies of a specific sequence of DNA present in the sample.
2. DNA amplification occurs by repeated thermal cycles
3. The number of copies of the specific sequence doubles after each cycle
4. After forty cycles, a single copy becomes around 2 trillion copies

Components of a PCR reaction
• DNA template (virus, bacterial or human gene)
• dNTP’s (a mix of all four nucleotides required to

build new DNA strands: A, T, C, G)
C
• Primers (oligonucleotides of about 20 nucleotides,
T T G G C
which will anneal to the target DNA)
A A
• Polymerase (natural thermostable Taq polymerase

that can function at an optimum temperature of C
C
about 70°C) G
T A Taq polymerase
G
• Buffer (Mg2+, Tris-HCl, Triton: provides the
optimal conditions for the polymerase to work)
The phases of PCR
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reserved - 301-8245,Rev
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November2020,
2020
The first phase of a PCR cycle - denaturation
separation of DNA strands
• 90-95 ºC
• 20-30 sec.

The second phase of a PCR cycle - annealing
specific primer binding
5’. Reverse primer
Temperature: 40-65 ºC
Duration: 20-40 seconds
5’ Forward primer

The third phase of a PCR cycle- extension
DNA strand synthesis
3’ 5’. Reverse primer
• 60-75 ºC
Duration depends
on the sequence size
to be amplified
Pol
3’
5’ Forward primer

Thermal profile of PCR cycling
1 1 1
1
1. Denaturation
3 3 3
3
2 2 2
2
Note: a PCR usually consists of 30 to 40 cycles

1 1 1
1 1 1
3 3 3 2. Annealing
3 3 3
2 2 2
2 2 2

1 1 1
1 1 1
3 3 3
3 3 3
2 2 2 3. Extension
2 2 2

Number of DNA copies obtained by PCR
‒ In theory, the number of copies of target DNA doubles with each

cycle, which means a PCR efficiency factor E = 2
• Starting concentration (0 cycles)= C0

• After one cycle: C0 x 2
• After 2 cycles: C0 x 4
• After 3 cycles: C0 x 8
• After n cycles: C0 x 2n
‒ In reality, this replication rate cannot be sustained forever and the

doubling becomes less than a doubling , then no replication at all
Factors influencing PCR efficiency
• Test design (Manufacturer)

‒ Primer/Probe design
‒ Type of DNA polymerase
‒ Quality of reagents
‒ Master mix
‒ PCR cycling conditions: temperatures and duration of the phases
• Pre-analytics (Laboratory technician)

‒ Quality of Reagents, due to transport and storage conditions
‒ Sample quality : presence of PCR inhibitors

Product detection at end point
In the classical PCR, the detection is performed at end-point

(end of PCR)
ladder
1. A mix of fragments of known sizes (ladder) is loaded into an agarose
gel, as reference, to calculate the size of the PCR products.
2. The PCR product is also loaded into the gel
3. An electric field is applied, so negatively charged molecules migrate

toward the positive pole
ladder PCR
4. The PCR product migrates according to size products
5. The DNA is stained using ethidium bromide, visible under UV lamp
6. If the target we are looking for is present in the sample, a PCR

product of the expected size is present

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Basics of

Polymerase Chain Reaction

1 © 2017-2020 Cepheid - All rights reserved - 301-8245, Rev B, November 2020,

Basics of Molecular Biology

PCR Basics Part III Definition of Melting Temperature

The General Objective of this Module Is to Give You an

At the end of the training, you will be able to:

• List the elements involved in the PCR Process

3 © 2017-2020 Cepheid - All rights reserved - 301-8245, Rev B, November 2020,

4 © 2017-2020 Cepheid - All rights reserved - 301-8245, Rev B, November 2020,

Genetic information contained in DNA

▪ DNA is built in a double helix

• Genetic information in bacteria is encoded in DNA

7 © 2017-2020 Cepheid - All rights reserved - 301-8245, Rev B, November 2020,

DNA is made up of 4 nucleotides

9 © 2017-2020 Cepheid - All rights reserved - 301-8245, Rev B, November 2020,

Most DNA is double stranded and pairs in a unique way:

10 © 2017-2020 Cepheid - All rights reserved - 301-8245, Rev B, November 2020,

• New DNA is made by enzymes called DNA

11 © 2017-2020 Cepheid - All rights reserved - 301-8245, Rev B, November 2020,

1. PCR (Polymerase Chain Reaction) is a chain reaction which generates

13 © 2017-2020 Cepheid - All rights reserved - 301-8245, Rev B, November 2020,

• dNTP’s (a mix of all four nucleotides required to

• Polymerase (natural thermostable Taq polymerase

16 © 2017-2020 Cepheid - All rights reserved - 301-8245, Rev B, November 2020,

5’. Reverse primer

17 © 2017-2020 Cepheid - All rights reserved - 301-8245, Rev B, November 2020,

3’ 5’. Reverse primer

18 © 2017-2020 Cepheid - All rights reserved - 301-8245, Rev B, November 2020,

Note: a PCR usually consists of 30 to 40 cycles

19 © 2017-2020 Cepheid - All rights reserved - 301-8245, Rev B, November 2020,

Note: a PCR usually consists of 30 to 40 cycles

20 © 2017-2020 Cepheid - All rights reserved - 301-8245, Rev B, November 2020,

Note: a PCR usually consists of 30 to 40 cycles

‒ In theory, the number of copies of target DNA doubles with each

• Starting concentration (0 cycles)= C0

‒ In reality, this replication rate cannot be sustained forever and the

• Test design (Manufacturer)

• Pre-analytics (Laboratory technician)

24 © 2017-2020 Cepheid - All rights reserved - 301-8245, Rev B, November 2020,

In the classical PCR, the detection is performed at end-point

2. The PCR product is also loaded into the gel

3. An electric field is applied, so negatively charged molecules migrate

5. The DNA is stained using ethidium bromide, visible under UV lamp

6. If the target we are looking for is present in the sample, a PCR

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