HALİÇ UNIVERSITY
FACULTY OF SCIENCE AND LITERATURE
DEPARTMENT OF MOLECULAR BIOLOGY AND GENETICS
2024-2025 ACADEMIC YEAR FALL SEMESTER
MOLECULAR BIOLOGY APPLICATIONS LABORATORY-I (MBG405-1)
Assist. Prof. Deniz KANCA DEMİRCİ
Laboratory Assistants
Hatice KURNAZ
Şafak ŞENER
Experiment 7 : Polymerase Chain Reaction (PCR) optimization
Ymen Mazhoud (21029980113)
Group members: Noura Portio Traore, Bita Rostami, Wael Ibrahim
Experiment date: 29/11/2024
Submission date: 06/12/2024
1. Aim: This experiment aims to optimize PCR conditions for the VDBP gene amplification
by evaluating the effects of MgCl2, primer concentration, DMSO concentration, and
DNA concentration on the amplification specificity and efficiency.
2. Introduction:
PCR is an essential molecular biology technique broadly used to amplify DNA
sequences. This enables a thorough analysis of the specific sequences and the ability to
use them in many scientific and medical fields [1]. During this experiment, the focus was
enhancing the PCR conditions to amplify the VDBP gene isolated from blood samples.
The main target parameters are MgCl2, primers, and DMSO concentrations [2]. Each
chemical used has a crucial role in the PCR amplification procedure.
The VDBP gene encodes the vitamin D-binding protein, this protein helps vitamin D
transport and immune system regulation. Diseases such as osteoporosis and autoimmune
disorders result from mutations in this gene, therefore studying and understanding the
mutations helps give valuable insights regarding potential treatments [2].
MgCl2, a cofactor for Taq DNA polymerase, enhances its enzymatic activity by
stabilizing enzyme-substrate interactions, facilitating nucleotide incorporation, and
maintaining the structural integrity of the enzyme-DNA complex. However, high MgCl2
concentrations can cause nonspecific amplification, while low levels may inhibit DNA
polymerase activity [1, 3]. Primers are short, sequence-specific oligonucleotides that bind
to specific DNA sequences and guide their synthesis. Their concentration affects the yield
of PCR products. Primer dimers or non-specific binding can result from inappropriate
primer concentrations in the sample [1]. DMSO improves amplification efficiency by
reducing secondary DNA structures, specifically in GC-rich DNA sequences. However,
excessive use of DMSO may inhibit polymerase activity [3].
Optimization of PCR parameters is crucial to avoid obtaining false positives or false
negatives, a false positive occurs when a genetic material or pathogen isn't there but the
test indicates its presence, while a false negative results when a genetic material or a
pathogen is present while the test fails to detect it, both false tests can lead to incorrect
diagnoses and gene identification. This can be very crucial in clinical diagnostics where
the correct detection of genetic mutations or pathogens is critical [4].
Additionally, the use of advanced PCR techniques such as hot-start DNA polymerases
and optimized buffers, has expanded its applications in areas of forensics and
personalized medicine [5]. This experiment's significance lies in optimizing these
variables to enhance PCR efficiency and reliability, which is crucial for downstream
applications such as biotechnology research, genetic studies, and diagnostics [1, 4].
3. Materials [2]:
Equipment:
- Eppendorf tubes
- Test tube rack
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- Micropipette
- Micropipette tips
- CD marker
- Electrophoresis equipment ( gel tank, Casting Tray, Comb, buffer chamber, casting
dams, tank lid, electric cables)
Chemicals:
- 10X Buffer
- MgCl₂
- dH₂O
- dNTPs
- Forward Primer
- Reverse Primer
- DMSO (Dimethyl sulfoxide)
- Taq Polymerase (DNA polymerase)
- Agarose Gel (0.7%)
- 6X Loading Dye (Thermo Fisher Scientific)
- 1kb GeneRuler DNA Marker (Thermo Fisher Scientific)
- Ethidium Bromide (10 mg/ml) (Thermo Fisher Scientific)
Devices:
- Thermal cycler
- Agarose gel electrophoresis system and power source
- UV transilluminator
Organic material:
- DNA (isolated from blood samples
4. Methods [2]:
The following tables are used as a guide during this experiment:
Table 1. Reaction contents to be prepared for DNA, MgCl2, primer, and DMSO
optimization
2
Table 2. master mix content
1. Prepare four different master mixes based on the specific variables: DNA, MgCl₂,
primers, and DMSO according to the volumes and chemicals listed in Table 1 and
Table 2. Add all required chemicals to the master mix tubes without adding DNA.
2. For each group, prepare a PCR reaction mix for 25 µl per tube by combining the
listed chemicals from the master mix. Make sure that the reactions are distributed
equally into the 5 PCR tubes of each group. Ensure that DNA is not added to the
negative control.
3. Program the PCR thermocycler with the following cycle conditions (temperature and
time):
- Initial Denaturation: 94°C for 5 minutes.
- Denaturation: 94°C for 45 seconds.
- Annealing: 60°C for 1 minute (for 35 cycles).
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- Extension: 72°C for 1 minute.
- Final Extension: 72°C for 10 minutes.
4. Prepare a 0.7% agarose gel and mix 1 µl PCR product from each tube with 5 µl of 6X
loading dye. Load the 6 µl of PCR tube product into the agarose gel wells. 1 kb
GeneRuler DNA Marker is loaded to the first well for reference. Run the gel at 120V
for 30 minutes.
5. Visualize the PCR products under UV light using a transilluminator and analyze the
results.
5. Results:
In this experiment, each group (4 groups) prepared five PCR reaction tubes
following the parameters listed in Tables 1 and 2. Our group was responsible for
preparing the master mix for the second tube, multiplying the volumes listed in
the tables by five to yield a total of 80 µl of master mix. 16 µl of this master mix
was distributed into each of the five experimental tubes. The primer concentration
was increased from tubes 1 to 4, according to the volumes specified in Table 2.
One microliter of DNA was added to each of the four experimental tubes, while
the 5th tube, the negative control contained no DNA. The final reaction volume for
all tubes was adjusted to 25 µl by adding the required volume of dH2O.
The wells in the agarose gel electrophoresis gel were loaded in the following
order, from left to right: the first well contained the DNA ladder, followed by the
four tubes from Group 1 (three samples and one negative control). Next were the
five tubes from Group 2, followed by five tubes from Group 3 and five tubes from
Group 4. No DNA bands were observed in any of the wells corresponding to
groups 1, 2, and 3. 4 thin bands, with smear were observed in the tubes
corresponding to group 4Figure 1 presents the image of the electrophoresis gel,
which shows the results from the PCR reaction.
Figure 1. Agarose gel electrophoresis results. DNA ladder in the first well,
followed by the samples from Group 1, Group 2, Group 3, and Group 4,
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6. Discussion:
In this experiment, the main goal was to identify the effect of each chemical on PCR
while keeping the other parameters constant. The results demonstrated that DNA bands
were only obtained in the agarose gel electrophoresis for the four tubes containing
increased DNA concentrations, belonging to the fourth group. This highlights the
importance of DNA template concentration in PCR amplification. On the other hand, the
complete absence of bands in all other three groups can be explained by several factors,
maınly involving a poor choice of PCR conditions or experimental errors caused by
wrong measurements or faulty devices. Low DNA concentration or degraded DNA can
hinder the PCR reaction by failing to reach the threshold necessary for detectable
amplification, causing no visible bands when observed under UV light.
The MgCl₂ concentration has a crucial role in PCR amplification. Mg² ⁺ ions are cheating
agents and cofactors for Taq DNA polymerase, they function by enhancing its enzymatic
activity through stabilization of the enzyme-template complex. Low Mg² ⁺ concentration
results in Taq Polymerase activity inhibition, insufficient Mg² ⁺ decreases nucleotide
binding efficiency, while excess Mg²⁺ increases non-specific binding and amplification
of unwanted DNA sequences. Therefore, optimization of MgCl₂ concentration is
essential for PCR amplification.
In addition, regarding primer concentration and annealing efficiency, Low primer
concentrations in certain reactions might have resulted in the lack of efficient annealing
to the DNA template, causing the failed amplification. In contrast, excessive primer
concentrations can cause primer dimer formation and non-specific binding to other DNA
regions, reducing PCR efficiency.
Regarding technical errors, pipetting inaccuracies due to faulty pipettes and small
volumes, contamination, or poor mixing of reagents, could also result the absence of
bands. Thermal cycler settings that are not suitable for the DNA polymerase used can also
be a reason for the failed amplification.
The presence of smear and thin bands in the group with increased DNA concentrations
suggests DNA degradation or primer dimer formation. Non-specific amplification can
also be seen.
Overall, this experiment highlights the importance of optimizing PCR conditions,
including DNA concentration, MgCl₂ levels, primer concentrations. Future
improvements by ensuring high-quality DNA templates and minimizing technical errors
would further enhance the PCR results.
7. References:
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[1] Mullis, K., Faloona, F. (1987). Specific synthesis of DNA in vitro via a
polymerase-catalyzed chain reaction. Methods in Enzymology, 155, 335-350.
[Link] Access date: 05/12/2024
[2] Haliç University Faculty of Science and Literature, Department of Molecular
Biology and Genetics. (2024). Molecular Biology Applications Laboratory-I
Manual: Experiment 7 - Polymerase Chain Reaction (PCR) optimization.
[3] Brownie, J., et al. (1997). The elimination of primer-dimer accumulation in
PCR. Nucleic Acids Research, 25(16), 3235-3241.
[Link]
[4] Dieffenbach, C. W., & Dveksler, G. S. (1995). PCR Primer: A Laboratory
Manual. Cold Spring Harbor Laboratory Press.
[5] Higuchi, R., Dollinger, G., Walsh, P. S., & Griffith, R. (1992). Simultaneous
amplification and detection of specific DNA sequences. Biotechnology, 10(4),
413-417. [Link] access date: 05/12/2024
