MLSMOLBL: MOLECULAR BIOLOGY (LABORATORY)

MODULE 1 (FINALS): POLYMERASE CHAIN REACTION

BS MEDICAL TECHNOLOGY 3ND TERM | S.Y. 2023-2024
LECTURER: Alvin John Erese TRANSCRIBED BY: TUTI
TITETITETETE

LEGEND 2.To characterize cells or organisms by

determining the DNA/RNA sequence of an
➢ From the Book
amplified DNA/RNA target
✷ MS Teams Notes
♛ Prof’s Side Notes ➢ Target amplification involves making many copies
♛ Own Side Notes of a specific DNA sequence. (Amplicons)
➢ PPT Notes ➢ PCR is DNA replication in vitro
IETETETETETTETETETET

MODULE OUTLINE ➢ DNA template must unwind and split up.

I. Introduction ● DNA Polymerase as main replication enzyme
II. Polymerase Chain Reaction ➢ Replication via BASE PAIRING RULE
a. Components of PCR
III. Typical PCR Set-up in Laboratory ➢ By amplifying the amount of nucleic acid materials
a. Pre-PCR
b. PCR obtained from clinical samples. Molecular
c. Post-PCR techniques can effectively provide a quantitative
IV. Modifications of Traditional PCR
a. Reverse Transcriptase PCR (RT-PCR) representation of an individual’s health status.
b. Multiplex PCR
c. Real-Time Quantitative PCR (qPCR)

INTRODUCTION
➢ Early analyses of nucleic acids were limited by the
availability of DNA.Generating enough copies of a
single gene required propagation of millions of cells
in culture or isolation of large amounts of genomic
DNA.
➢ The first specific amplification method of any type
was the polymerase chain reaction (PCR).
➢ PCR is the first and prototypical method for
amplifying target nucleic acid. WHAT ARE THE COMPONENTS OF PCR
➢ Other amplification methods have been developed
Medium for all other components,
based on making modifications to PCR.
specially purified, double distilled,
Water deionized,autoclaved, nuclease free, and
POLYMERASE CHAIN REACTION does not contain detectable amounts of
nucleic acid
➢ Developed by Kary Mullis in mid-1980’s, granted
Nobel prize in 1993. Stabilizes the DNA polymerase, DNA and
Buffer
➢ A “copy machine” for DNA which revolutionized nucleotides

molecular biology. DNA Template Contains the target region of interest

➢ PCR allows the rapid synthesis of designated
Thermostable Such as Taq polymerase, to catalyze
fragments of DNA Polymerase DNA-dependent DNA synthesis
➢ In diagnostic clinical laboratories, this technique is
primarily used for the following purposes:
1. To determine and confirm cellular or
organismal presence by the detection of its
genomic DNA or RNA (Bacterial, Viral,
Eukaryotic organisms)
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 PRE-PCR
● Oligodeoxynucleotides that flank the
region of interest ➢ First step in PCR analysis
➢ Prior to performing Molecular Biology techniques.
● Forward:
○ Locates the replication start point Sample treatment requires cellular lysis and DNA
along the template’s sense strand or RNA extraction.
Primer Pair
● Reverse:
➢ Once the nucleic acid material is extracted and
○ Marks a second replication start
point along the antisense, removed of impurities targeted amplification
downstream of the forward primer techniques and other sequencing techniques may
● Length is approximately 18-30 base be done.
nucleotide
PCR
● Cofactor for polymerase activity 1. Heat denaturation: High heat to disrupt and
● Stabilizes the DNA double-helix separates DNA duplexes
● Too little:
2. Primer annealing: attachment of primers to the
Magnesium ○ Enzyme won’t work. template strand
(Mg2+)
3. Primer extension: elongation of daughter strand
● Too much: with DNA Polymerase
○ DNA extra stable,
○ Non-specific priming

Deoxynucleotides
Triphosphates Equimolar building blocks of DNA
(dNTPs)

Typical PCR Set-up in Laboratory

Pre-PCR PCR Post-PCR

● Isolation of Nucleic
Acid from samples
● Preparation of PCR
Reaction Thermal Cycling
Components (Denaturation, Analysis of
(Mastermix) Annealing, Amplified DNA
● • Addition of Nucleic Extension)
Acid Template into
the PCR
● Reaction Mastermix ➢ The cycle would continue repetitively in a
predetermined turnaround time. As the cycle
repeats, more copies of nucleic acid materials are
made in the polymerase chain reaction.
➢ Running the correct controls during every PCR run
is essential for ensuring

CONTROLS FOR PCR

Positive controls ensure that the enzyme is

Positive active, the buffer is optimal, the primers are
Control priming the right sequences, and the thermal
cycler is cycling

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 2. Multiplex PCR
A negative control without DNA (also called a
Negative contamination control or reagent blank ) ensures ➢ Multiplex PCR is a powerful technique that
Control that the reaction mix is not contaminated with enables amplification of two or more products in
without DNA template DNA or amplified products from a
parallel in a single reaction tube. It typically
previous run.
employs different primer or probe pairs in the
A negative control with DNA that lacks the target same reaction for simultaneous amplification of
Negative sequence (negative template control) ensures
Control multiple targets.
with DNA that the primers are not annealing to nontarget
sequences of DNA. ➢ Multiplex PCR is used widely in genotyping
applications and multiple areas of DNA testing in
POST-PCR research, forensic, and diagnostic laboratories.
➢ After PCR, a variety of methods can be used to
analyze the product. Most commonly, the PCR
product is analyzed by gel or capillary
electrophoresis. Depending on the application, the
size, presence, or intensity of PCR products is
observed after electrophoresis.
➢ Analyzing PCR Products
1. Gel electrophoresis
2. Capillary electrophoresis
3. Real Time or Quantitative PCR (qPCR)
3. Nucleic acid hybridization
➢ For some situations, though, the clinician is also
4. DNA sequencing
interested in how much of the target sequence is
5. Real-time quantitative PCR present.
➢ In real-time PCR, the accumulation of amplification
MODIFICATIONS OF TRADITIONAL PCR
product is measured as the reaction progresses, in
1. Reverse transcriptase PCR (RT-PCR) real time, with product quantification after each
2. Multiplex PCR cycle.
3. Real-time quantitative PCR ➢ Detectable fluorescence in earlier cycles of the
1. Reverse Transcriptase PCR amplification program indicates higher amounts of
starting template, whereas fluorescence
➢ Refers to the conversion of the RNA template into
appearing in later cycles indicates lower amounts
its complementary DNA strand (cDNA) is an
of starting template.
essential step in the analysis of gene transcripts
cDNA can be sequenced, cloned and applied to ➢ The PCR cycle at which sample fluorescence
estimate the copy number of specific genes in crosses the threshold is the threshold cycle, or CT.
order to characterize and to validate gene ➢ Real-time detection of PCR products is enabled by
expression the inclusion of a fluorescent reporter molecule in
each reaction well that yields increased
fluorescence with an increasing amount of
product DNA.
➢ The first approach to qPCR utilized EtBr, which is
specific to double-stranded DNA. Dye is still used
for routine qPCR, except that EtBr has been
replaced by SYBR green, another dye specific to
double-stranded DNA.
➢ The advantage of SYBR green is its specificity and
robust fluorescence comparable to that of EtBr
and its reduced toxicity. Both dyes bind and
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 fluoresce specifically in the double-stranded DNA
product of the PCR
➢ The purpose of the probe is to identify one or
more sequences of interest within a large amount
of nucleic acid. The probe therefore should
hybridize specifically with the target DNA or RNA
that is to be analyzed. The probe can be RNA,
denatured DNA, or other modified nucleic acids.
➢ Real-time PCR results can either be qualitative
(the presence or absence of a sequence) or
quantitative (copy number).
These are the Common Probes used in PCR

TaqMan was developed from one of the first

TaqMan probe-based systems for quantifying cDNA
by qPCR.

Molecular Measures the accumulation of product at the

Beacons annealing step in the PCR cycle.

A target-specific primers which are tailed at

the 5 ′ end with a sequence complementary to
Scorpions part of the internal primer sequence, a
quencher, a stem-loop structure, and a 5 ′
fluorophore

Fluorescent Utilizes two specific probes, one with a 3′

Resonance fluorophore (acceptor) and the other with a 5′
Energy Transfer
(FRET) catalyst for the fluorescence (donor)

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