COLLEGE OF NATURAL SCIENCES

DEPARTMENT OF BIOCHEMISTRY AND

SPORTS SCIENCES

Experiment on the Amplification of DNA using Polymerase Chain

Reaction (PCR) and visualization by Agarose Gel Electrophoresis

NAME: ANZO SIMON

REG NO: 22/U/5784
YEAR: 2
SEMESTER: 2

DATE OF PRACTICAL: 4TH FEBRUARY, 2024

DATE OF SUBMISSION: 2nd APRIL, 2024

PARTNERS.
NAMES REG. NO.
1. Ampire Arnold 22/U/5765
2. Balyawo Hope 22/U/5870

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ABSTRACT
The Polymerase Chain Reaction (PCR) is a technique for amplifying specific DNA sequences,
it is often used alongside agarose gel electrophoresis for analysis of the PCR products. The
main aim of the experiment focuses on effective amplification DNA samples following its
extraction using the PCR method followed by visualization by gel electrophoresis using
Ethidium bromide as a stain in a bid to validate the efficacy of the PCR in exponentially
amplifying target sequences as well as to assess the reproducibility of results.

PCR was performed effectively basing on the ability of DNA polymerase to synthesize new
strand of DNA complementary to the offered template strand with the primers targeting a
defined region of sample DNA.

Temperature cycling that occurs in three stages involving denaturation, annealing and
elongation which are repeated several times doubling the DNA copies produced every time in
a thermal cycler. It is also revealed that positive controls using known templates confirmed
successful amplification, while negative controls lacking template showed no contamination.
The DNA polymerase enzyme synthesizes new DNA strands by adding complementary
nucleotides to the 3' end of the primers, extending the DNA sequence in the 5' to 3' direction.
To amplify DNA, a PCR master mix was prepared with different components. It was then
added into a microfuge tube mixed gently with a vortex, and then centrifuged. Resultant
mixture was added in equal volumes to 30 PCR tubes followed by a template, and then thermal
cycled in a PCR machine.

Visualization of amplified DNA was evaluated by agarose gel electrophoresis and Ethidium
bromide staining. Agarose gel electrophoresis separated the PCR products by size. Under UV
illumination, distinct bands representing the amplified target were visualized in lanes loaded
with experimental samples. However, minor variability in band sizes was noted across
experimental replicates despite efforts to standardize PCR conditions. Results for both samples
and standard were red bands of variable size under UV illumination.

The standard showed prominent band of expected size corresponding to the amount of DNA.
However, the bands corresponding to the samples were smaller and not clearly visible
indicating for less DNA availability in the samples hence indicating that the amplification
process was not efficient enough to extract the required amount of DNA in the samples. This
could be due to contamination or DNA degradation.

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INTRODUCTION
PCR follows the event of DNA extraction which basically refers to the process of isolating
DNA from biological samples including cells, tissues, blood, saliva, or environmental sample
where the extracted DNA is purified and later serves as the template for amplification.

Polymerase chain reaction (PCR) is defined as a laboratory technique used for rapidly
producing (amplifying) millions to billions of copies of a specific segment of DNA, which can
then be studied in greater detail.(Erlich, 1989)

PCR involves using short synthetic DNA fragments called primers to select a segment of the
genome to be amplified, and then multiple rounds of DNA synthesis to amplify that segment.

The technique is applied in early stages of processing DNA for sequencing or when generating
forensic DNA profiles from tiny amounts of DNA. It can also be used to detect the presence or
absence of a gene to help identify pathogens during infection with a prominent example being
during the Covid-19 pandemic where the PCR tests were used to detect whether a person’s
swab sample had a segment of the virus’s genetic material, to provide a positive or negative
infection result.(Zhu et al., 2020)

The PCR is typically a primer mediated enzymatic amplification of DNA, it uses the ability of
DNA polymerase to synthesize new strand of DNA complementary to the offered template
strand. Primer is needed because DNA polymerase can add a nucleotide only onto a preexisting
3'-OH group to add the first nucleotide. DNA polymerase then elongate its 3’ end by adding
more nucleotides to generate an extended region of double stranded DNA.

Important components such as DNA Template which is the double stranded DNA (dsDNA) of
interest, separated from the sample, DNA polymerase which is a thermostable polymerase that
does not rapidly denature at high temperatures (98"), and can function at a temperature
optimum of about 70°C, Oligonucleotide primers which are short pieces of single stranded
DNA (often 20-30 base pairs) which are complementary to the 3' ends of the sense and anti-
sense strands of the target sequence, Deoxy nucleotide triphosphates: Single units of the bases
A, T, G, and C (dATP, АТТР, dGTP, dCTP) that provide the energy for polymerization and
the building blocks for DNA synthesis. Buffer system which Includes magnesium and
potassium to provide the optimal conditions for DNA denaturation and renaturation; also
important for polymerase activity, stability and fidelity.

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The amplification process occurs involving three stages including; the denaturation step, where
the double-stranded DNA template is heated to a high temperature (usually around 94-98°C).
This causes the DNA strands to separate and unwind, breaking the hydrogen bonds between
the complementary base pairs. As a result, the DNA is converted into single-stranded DNA
molecules.

After denaturation, the reaction temperature is lowered (typically to around 50-65°C). During
this step, short DNA sequences called primers anneal to the complementary sequences on each
of the single-stranded DNA molecules. Primers are designed to flank the region of DNA that
needs to be amplified. They provide the starting point for DNA synthesis by DNA polymerase.

Once the primers are annealed, the reaction temperature is raised to the optimal operating
temperature of a DNA polymerase enzyme (usually around 72°C). This enzyme synthesizes
new DNA strands by adding complementary nucleotides onto the primers, extending them
along the DNA template. DNA polymerase reads the template strand and adds nucleotides in a
5' to 3' direction. This step effectively amplifies the targeted DNA sequence.(van Pelt-Verkuil
et al., 2008)

The denaturation, annealing, and extension steps constitute one cycle of PCR. The entire
process is typically repeated for 20 to 40 cycles, depending on the desired amount of DNA
amplification. Each cycle doubles the number of DNA molecules, resulting in an exponential
increase in the number of copies of the target DNA sequence.(Marot et al., 2021)

After successful completion of PCR, electrophoresis is often used to validate the efficacy and
check the quantity and size of the DNA fragments produced.

PCR is applied in different fields including medicine where it can be used for genetic testing
for presence of genetic disease mutations such as hemoglobinopathies, cystic fibrosis, and other
inborn errors of metabolism.(Jeffery et al., 1997)

It can also be used for the detection of disease-causing genes in suspected parents who act as
carriers, studying of alteration to oncogenes may help in customization of therapy and can also
be used as part of a sensitive test for tissue typing, vital to organ transplantation genotyping of
embryo also helps to monitor the gene in gene therapy.

PCR is applied in Forensics where it can be used as a tool in genetic fingerprinting. This
technology can identify any one person from millions of others in case of crime scene, rule out

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suspects during police investigation, paternity testing even in case of availability of very small
number of specimens.(Kadri, 2019)

OBJECTIVES.
The aims of the experiment are indicated as below;
To amplify DNA using the PCR technique
To visualize the amplified DNA by using agarose gel electrophoresis.

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PROCEDURES
Requirements
Materials and apparatus
Master Mix. Ethidium bromide
PCR Water. Template DNA.
10x PCR buffer. 25mM MgCl2.
10mM Deoxynucleotide mix. Microfuge tube.
10μMForward primer. Centrifuge.
10μMReverse primer. 30 PCR tubes.
Taq DNA polymerase. PCR machine.
Agarose gel Gel electrophoretic tank.

Procedure for DNA amplification (PCR)

Mater mix was prepared in a 200μl microfuge tube comprising of a mixture of different
components including water, 10x PCR buffer, and 10mM Deoxynucleotide Mix, 10μM
forward primer, 10μM reverse primer, Taq DNA polymerase, template DNA, 25mM MgCl2.

All the components were added in the order as shown in the table below
Component Final concentration Total volume (ml)
1 10x PCR Buffer 1x 27.5
2 10mM Deoxynucleotide Mix 200µM 0.875
3 10µM Forward primer 0.1µM 2.75
4 10µM Reverse primer 0.1µM 2.75
5 Taq DNA polymerase 0.05units/µL 2.75
6 Template DNA (typically 10ng) 200pg/µL -
7 25mM MgCl2 0.1mM -
8 Water 73.56

The contents were mixed, and briefly centrifuged to collect the components to the bottom of
the tube. Equal volumes of the Master Mix were added into each of the 30 PCR tubes followed
by the template. 50μL of mineral oil were added to the top of each PCR tube to prevent
evaporation. The PCR tubes were placed in the Thermocycler and the machine programed with
desired parameters which vary depending on the primers and thermal cycler used.

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Procedure for the agarose gel electrophoresis.
1g of agarose was weighed into 100ml volumetric flask. 100ml of TAE buffer was added to
the agarose in the flask. The mixture was then heated on a hot plate for 2 minutes, 2-5μl of
Ethidium bromide were added while still hot and swirled gently to mix the contents and was
allowed to cool for 8 minutes at room temperature.

A comb was placed in the gel caster, and the agarose gel was poured into while still hot and
allowed to set at room temperature. The comb was pulled out carefully after settling and the
gel was placed into the electrophoretic tank filled with 1XTAE buffer. Taking into account the
order, 2-5μl of the each of the standards as well the samples were loaded after mixing with 2-
5μl of the DNA loading buffer into the wells.

The tank was then connected to a DC supply and allowed to run until the indicator dye had
moved about 1cm to the opposite end of the gel. At point, the DC supply was disconnected
from the tank and the gel carefully removed from the tank and observed under UV. A photo of
the gel was taken as recorded in the results section.

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RESULTS

Figure showing the bands as seen under UV light

Positive control band: A band at the expected size for the target DNA sequence was attained
in the standard. This confirms that the PCR reaction was successful and that the primers were
able to amplify the target sequence validating successful PCR amplification and efficacy of the
primers.

Negative control: No band should be visible in the negative control reaction, which contains
no template DNA. This confirms that there was no contamination in the PCR reaction
validating the integrity of the results.

Samples: Red bands for the target DNA sequence were observed. Size of the bands varied; the
bands were however small. This size corresponds to the amplified DNA fragments in the
experimental samples which were visualized. The smaller, fainter bands in samples imply
lower abundance of amplified DNA compared to the positive control.

Potential reasons for inconsistent yields include ineffective primer annealing, insufficient
template DNA, or inadequate amplification efficiency.

Further optimization of parameters like primer design, template quantity, thermo cycling
conditions, and reagent concentrations may improve uniformity.

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DISCUSSION
Polymerase Chain Reaction (PCR) is a widely used technique in molecular biology for
amplifying a specific segment of DNA. The principle behind PCR is based on the ability of a
DNA polymerase enzyme to synthesize new DNA strands complementary to a template strand
of DNA. For DNA to be amplified, it under goes a couple of events in a PCR which include
Denaturation, annealing and elongation. Denaturation is the first step and involves heating of
the DNA sample to a high temperature (around 94-98°C), which causes the double-stranded
DNA to denature or separate into two single strands. Annealing is the second event and
involves lowering of temperature to allow short DNA primers to renature by binding to their
complementary sequences on the single-stranded DNA template. These primers mark the start
and end points of the DNA segment to be amplified. Elongation is the last event where the
temperature is raised again, and a DNA polymerase enzyme synthesizes new DNA strands by
adding complementary nucleotides to the 3' end of the primers, extending the DNA sequence
in the 5' to 3' direction. The polymerase enzyme used in PCR is typically a thermostable enzyme
(e.g., Taq polymerase) that can withstand the high temperatures used in the denaturation step.

These three stages were repeated 20-40 times, doubling the number of DNA copies each time.
This is called thermal cycling, which was carried out by a thermal cycler machine. By repeating
these steps in a cyclic manner, each cycle of PCR doubles the number of DNA molecules,
leading to exponential amplification of the target DNA segment.

After a sufficient number of cycles, there are millions of copies of the target DNA segment,
which can then be used for various downstream applications, such as DNA sequencing,
cloning, and genetic analysis.

The amplified DNA copies were further visualized in agarose gel electrophoresis technique
under Ethidium bromide. Ethidium bromide is very sensitive and can detect small amounts of
DNA.

It binds to DNA by intercalating between the base pairs, causing the DNA to fluoresce when
exposed to UV light. The Ethidium bromide-DNA complex fluoresces orange-red, making the
DNA bands visible against the background of the agarose gel. Ethidium bromide-stained DNA
can be photographed using UV trans-illumination, allowing researchers to document and
analyze the results of agarose gel electrophoresis.

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Overall, the presence or absence of bands, their intensity, and their size relative to the expected
size provide details about the success of the PCR reaction and the presence of the target DNA
sequence in the samples. However, the results from this amplification were positive though
very lower than expected in the samples. Bands obtained were very small almost not visible as
seen from the results section. As compared to the standard, its bands were clearly defined easily
visible.

This indicated that there is lower abundance of amplified DNA available in the samples after
amplification. The DNA concentration could have been too low to be detected by ethidium
bromide staining which was used for visualization. The amplification could have been not
efficient to extract the required amount, or the initial template DNA concentration may have
been very low.

Other errors such as excess salts, residual phenol/chloroform from DNA extraction, or other
contaminants may have interfered with the amplification process, leading to low DNA yield.

Contamination with nucleases or other PCR inhibitors can lead to degradation or loss of DNA
during or after amplification. PCR is very sensitive to contamination, which can lead to false-
positive results or the amplification of unintended DNA fragments thus to prevent
contamination perform pre-PCR (sample preparation) and post-PCR (amplicon analysis) steps
in separate areas to prevent contamination.

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CONCLUSION
PCR is a very important tool in microbiology for amplification of DNA for downstream
applications, such gene sequencing, cloning, and analysis. Inefficient amplification by PCR
can negatively impact downstream applications by limiting the amount of DNA available,
reducing sensitivity, increasing background noise, impairing sequencing, and complicating
downstream analysis. This study demonstrated that Polymerase Chain Reaction (PCR) can
successfully amplify targeted DNA sequences when coupled with gel electrophoresis for
product analysis. The amplified DNA was subjected for visualization under gel electrophoresis
using ethidium bromide for staining, orange-red bands of DNA were attained whose size
corresponds to the amount of DNA present in the samples. The positive and negative controls
served as critical quality assurance measures, validating the efficacy of amplification and lack
of contamination. While DNA bands were detected across experimental samples, variability in
band intensity and size points to opportunities for optimizing PCR conditions to achieve more
consistent, uniform yields.

RECOMMENDATION
Errors in handling of reagents, and equipment used in the experiment could have led to
contamination of the samples. Such errors can be minimized by cleaning up the work space
before handling, and performing the PCR steps, use of more sensitive DNA staining method,
such as SYBR Safe or Gel Red which may detect lower DNA concentrations than ethidium
bromide.

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REFERENCES
1. Erlich, H. A. (1989). PCR technology (Vol. 246). Springer.

2. Jeffery, K. J., Read, S. J., Peto, T. E., Mayon-White, R. T., & Bangham, C. R. (1997).

Diagnosis of viral infections of the central nervous system: Clinical interpretation of

PCR results. The Lancet, 349(9048), 313–317.

3. Kadri, K. (2019). Polymerase chain reaction (PCR): Principle and applications.

Synthetic Biology-New Interdisciplinary Science, 1–17.

4. Marot, S., Calvez, V., Louet, M., Marcelin, A.-G., & Burrel, S. (2021). Interpretation

of SARS-CoV-2 replication according to RT-PCR crossing threshold value. Clinical

Microbiology and Infection, 27(7), 1056–1057.

5. van Pelt-Verkuil, E., Van Belkum, A., & Hays, J. P. (2008). Principles and technical

aspects of PCR amplification. Springer Science & Business Media.

6. Zhu, H., Zhang, H., Xu, Y., Laššáková, S., Korabečná, M., & Neužil, P. (2020). PCR

past, present and future. Biotechniques, 69(4), 317–325.

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