MTPC 140: Molecular Biology

and Diagnostics
MAVERICK V. SUSTIGUER, RMT
Senior Instructor
Medical Technology Department
Notre Dame of Marbel University
POLYMERASE
CHAIN
REACTION
LEARNING OUTCOMES:
At the end of the class discussion, the students should be
able to:
a. Define the basic terms associated to polymerase chain
reaction;
b. Elucidate the working principle of a standard PCR;
c. List and discuss examples of modifications of PCR, and
d. Enumerate the basic applications of PCR in medicine.
Polymerase Chain Reaction
• Developed by Kary Mullis (Nobel Prize in Chemistry)

• Allows amplification of a specific section of DNA from a

DNA template, generating millions of copies of the DNA
fragment

• Commonly used in research, forensic, and clinical laboratories

for a wide variety of applications including cloning, mutagenesis,
genotyping, and diagnosis of diseases.

Technique used to amplify an amount of nucleic acid in sample, probe

AMPLIFICATION

or signal to detect small amounts of nucleic acid

Target amplification: target sequence (PCR)
Probe amplification: probe bounded to the target (ligase-chain
reaction)
Signal amplification: signal to detect the target (bDNA and HCA)

14/04/2021 Maverick V. Sustiguer, RMT 4

 Workflow in Molecular Biology Laboratory

Sample Nucleic Acid Nucleic Acid

Nucleic Acid
collection and Extraction Detection
Amplification
Processing

14/04/2021 Maverick V. Sustiguer, RMT 5

PCR and Molecular Diagnostics
• The evolution of PCR completely transformed
medical diagnosis.
• Variations of PCR methods, such as Reverse
Transcription PCR (RT-PCR) and Real-Time PCR
(qPCR), have been developed to efficiently amplify
and quantitatively analyze DNA or RNA from
biological specimens.
• Novel PCR-based diagnostic tests allow detection
of infectious diseases, genetic variations and
mutations, cancer biomarkers, and blood disorders
with faster turnaround time and vastly improved
sensitivity and accuracy.

14/04/2021 Maverick V. Sustiguer, RMT 6

 • Thermocycler
Principle of PCR • Amplicon

Annealing Extension/
Denaturation
Elongation

• Melting • Anneal • DNA polymerase

• Melting temperature • Oligonucleotides
• dsDNA • Primer: determines specificity
• ssDNA • DNA synthesizer
14/04/2021 Maverick V. Sustiguer, RMT 7
14/04/2021 Maverick V. Sustiguer, RMT 8
 POLYMERASE CHAIN REACTION
dsDNA separates into two ssDNA (template)
90-96℃ for In vivo: helicase
DENATURATION
20-60 sec In vitro: heat energy *disruption of the hydrogen bonding and
hydrophobic stacking
Primers attach to both template strands (target sequence)
50-70℃ for
ANNEALING/ by binding with complementary bases on 3’ end of region to
20-90 sec
HYBRIDIZATION* be amplified
Temperature has to be lowered: Tm of the primer
Addition of nucleotides to the primers by DNA polymerase
Addition of complementary dNTPs to the template DNA
68-75℃ for
EXTENSION/ Thermus aquaticus: Taq DNA polymerase (1000 bases in 1
10-60 sec
ELONGATION minute)
Dependent to DNA polymerase and length of the target DNA
sequence
14/04/2021 Maverick V. Sustiguer, RMT 9
 POLYMERASE CHAIN REACTION
dsDNA separates into two ssDNA (template)
90-96℃ for In vivo: helicase
DENATURATION
20-60 sec In vitro: heat energy *disruption of the hydrogen bonding and
hydrophobic stacking

14/04/2021 Maverick V. Sustiguer, RMT 10

 POLYMERASE CHAIN REACTION
Primers attach to both template strands (target sequence)
50-70℃ for
ANNEALING/ by binding with complementary bases on 3’ end of region to
20-90 sec
HYBRIDIZATION* be amplified
Temperature has to be lowered: Tm of the primer

14/04/2021 Maverick V. Sustiguer, RMT 11

 POLYMERASE CHAIN REACTION
Addition of nucleotides to the primers by DNA polymerase
Addition of complementary dNTPs to the template DNA
68-75℃ for
EXTENSION/ Thermus aquaticus: Taq DNA polymerase (1000 bases in 1
10-60 sec
ELONGATION minute)
Dependent to DNA polymerase and length of the target DNA
sequence

14/04/2021 Maverick V. Sustiguer, RMT 12

PCR Cycling
1. Initial Denaturation
2. Denaturation
3. Annealing
4. Extension
5. Final Extension

14/04/2021 Maverick V. Sustiguer, RMT 13

REVERSE TRANSCRIPTION PCR (RT-PCR)
One-Step RT-PCR: The reverse
• PCR relies on the ability of transcription occurs in the same tube as the PCR
DNA polymerase to amplification.
synthesize a new DNA
Advantages: simple and fast setup, high
strand from a DNA
reproducibility, lower possibility of contamination
template.
• RNA: nucleic acid of
interest (mRNA → cDNA) Two-step RT-PCR: The reverse
*reverse transcription transcription and the PCR amplification occurs in
• Sensitive and commonly separate tubes.
used to detect and quantify Advantages: Multiple PCRs from one RT
messenger RNA reaction, flexibility for primers and PCR
conditions and long-term storage of cDNA
14/04/2021 Maverick V. Sustiguer, RMT 14
 One-Step RT-PCR Two-Step RT-PCR

14/04/2021 Maverick V. Sustiguer, RMT 15

 Analysis of PCR Products
• Amplified products are traditionally
detected by gel electrophoresis
after PCR cycling is completed.
• The PCR products resolved on the gel
are visualized by applying a stain such
as ethidium bromide.
• Standard PCR provides only a semi-
quantitative estimation of the
abundance of the PCR product.

14/04/2021 Maverick V. Sustiguer, RMT 16

Real-Time PCR or Quantitative PCR (qPCR)
• Variation of PCR developed
to quantitatively determine
the amount of target DNA
or cDNA nucleic acid.
• The same principle of
traditional PCR amplification
is applied
• PCR products are detected at
each cycle in "real-time"
using a thermocycler with
fluorescence detection
capability.

14/04/2021 Maverick V. Sustiguer, RMT 17

14/04/2021 Maverick V. Sustiguer, RMT 18
14/04/2021 Maverick V. Sustiguer, RMT 19
15/04/2021 Maverick V. Sustiguer, RMT 20
14/04/2021 Maverick V. Sustiguer, RMT 21
14/04/2021 Maverick V. Sustiguer, RMT 22
Monitoring DNA Amplification in Real-Time PCR
Utilizes a dye that emits fluorescence when
Fluorescent Dye-
incorporated into dsDNA
Based Detection
SYBR Green I
Utilizes fluorescently labeled oligonucleotide
Fluorescent Probe-
probes to detect specific DNA sequences
Based Detection
Taqman probe and FRET probe

15/04/2021 Maverick V. Sustiguer, RMT 23

14/04/2021 Maverick V. Sustiguer, RMT 24
Quantification of Nucleic Acid by Real-Time PCR
Absolute Quantification
The amount of the target nucleic acid is
reported as a copy number or a
concentration.
Relative Quantification
The amount of the target nucleic acid is
reported comparative to the abundance of
another gene.

15/04/2021 Maverick V. Sustiguer, RMT 25

 No template control - reaction with no DNA template input, which allows
the identification of nucleic acid contamination in the reagents.
No reverse transcriptase control - reaction with no reverse transcriptase
Negative
added, which allows detection of specific contaminating DNA in RNA
Controls
samples.
No amplification control - reaction with no DNA polymerase added,
which allows observation of background fluorescence that is not a result of
PCR amplification.
Used to demonstrate that the PCR conditions, including the primers used,
Positive are suitable for the analysis.
Control
Serial dilutions can be used to generate a standard curve for quantification.

14/04/2021 Maverick V. Sustiguer, RMT 26