HALİÇ UNIVERSITY

FACULTY OF SCIENCE AND LITERATURE

DEPARTMENT OF MOLECULAR BIOLOGY AND GENETICS

MOLECULAR BIOLOGY APPLICATIONS LABORATORY-I (MBG405-1)

Assist. Prof. Deniz KANCA DEMİRCİ

Laboratory Assistants
Hatice KURNAZ
Şafak ŞENER
Experiment 8: PCR Optimization with Touchdown PCR
Ymen Mazhoud (21029980113)
Group members: Noura Portio Traore, Bita Rostami, Wael Ibrahim
Experiment date:06/12/2024
Submission date: 13/12/2024

1. Aim:
 This experiment aims to optimize PCR conditions to avoid non-specific amplification
and/or primer dimer formation, using touchdown PCR and gradient PCR to identify the
optimal PCR conditions.
2. Introduction:

PCR is an anchor technique in molecular biology used for amplifying DNA sequences
[1]. Touchdown PCR and Gradient PCR are two optimization techniques that stand out
due to their capacity to enhance specificity and yield [2].
Touchdown PCR starts with an initial phase of relatively high annealing temperatures,
which are gradually lowered over the cycles. This step aims to increase primer-template
specificity by lowering non-specific binding and primer dimer formation [3]. By starting
with extreme conditions, the desired target amplification is favored over random
segments, ensuring higher specificity for subsequent amplification [4].
Alternatively, Gradient PCR employs a thermal cycler capable of altering temperatures
across rows and columns. Figure 1. Demonstrates a typical Gradient PCR setup and its
mode of function.

Figure 1. Gradient PCR temperature Setup. the temperature increases with the X and Y axes in
this example [8].
This setup enables simultaneous testing of multiple annealing temperatures while
keeping the other parameters constant, once the amplified samples are visualized using
Gel electrophoresis, an optimum temperature range can be selected [5]. Gradient PCR is
particularly useful and beneficial for optimizing new primers or protocols, it helps find
ideal amplification conditions [6].
These techniques address common PCR challenges faced during protocols. Touchdown
PCR is mainly used to reduce background amplification or noise, while Gradient PCR
can be considered a shortcut for experimental conditions optimization [7].
The experiment utilizes both methods, the goal is to highlight their comparative efficacy
in achieving high specificity and efficiency.
3. Materials [1]:

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 Equipment:
- Eppendorf tubes
- Test tube rack
- Micropipettes
- Micropipette tips
- CD marker
- Electrophoresis equipment ( gel tank, Casting Tray, Comb, buffer chamber, casting
dams, tank lid, electric cables)
Devices:
- Thermal cyclers:
Techne Gradient Thermal Cycler PZR device
Techne TC312 Thermal Cycler PZR device
- Agarose gel electrophoresis system and power source
- UV transilluminator
Chemicals:
- Taq Polm (5 U/μl)
- 10X PCR buffer
- MgCl2 (25mM)
- Cyt-R primer (5μM)
- Cyt-F primer (5μM)
- dNTP mix (each 10 mM)
- ddH2O
- 1 Kbp Marker
- DNA loading dye
- Agarose Gel (2%)
- Ethidium Bromide (10 mg/ml) (Thermo Fisher Scientific)
Biological material:
- Genomic DNA:

4. Methods [1]:
Reference tables:

Table 1. PCR tube mixture for each group in µL

Table 2. PCR tube arrangement in the 96-well gradient PCR thermal cycler

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1- 5 tubes per group will be prepared following the volumes given in Table 1. A master
mix containing the total volume of the 5 tubes’ chemicals is prepared. The volume of
each chemical is multiplied by 5 and added to the master mix tube. No DNA should
be added during this step.
2- Each group must add MgCl2, the only variable in this experiment, respectively to
what is stated in the table.
3- Once 5 tubes of equal volumes and chemical composition are prepared (24 µL), 1 µL
of DNA is added to 4 tubes, and the 5th tube is used as a negative control.
4- of the 4 tubes containing DNA from each group are placed in the Techne Gradient
Thermal Cycler PZR device. The order of placement must follow the instructions given
in Table 2. The settings listed below are used for this amplification procedure:
94°C for 5 minutes

94°C for 45 seconds

55–65°C for 45 seconds (30 cycles)

72°C for 1 minute

72°C for 5 minutes

5- The 4th and 5th tubes undergo touchdown PCR following the given conditions and
temperatures using Techne TC312 Thermal Cycler PZR device:
94°C for 5 minutes

94°C for 15 seconds

72°C for 1 minute

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 94°C for 15 seconds

60°C for 30 seconds

72°C for 5 minutes

6- Once the amplification process is complete, the samples are run on 2% agarose gel
electrophoresis for 30 minutes (120V). The gel is then visualized under UV light and
the results are analyzed and interpretations are made accordingly.

5. Results:
After amplifying the genomic DNA using touchdown PCR and Gradient PCR. The
following agarose gel electrophoresis obtained are observed in Figure 2.

Figure 2. Agarose gel electrophoresis results: The upper row shows gradient PCR, and
the Lower row shows touch-down PCR

No visible DNA bands were seen in any of the wells after amplification. The only visible
bands are the DNA ladder bands.

6. Discussion:

In this experiment, the objective was to enhance PCR conditions using two optimization
tools, Touchdown PCR and Gradient PCR to minimize non-specific amplification of the
target DNA sequence and primer dimer formation.

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The agarose Gel electrophoresis results showed no visible DNA bands in any of the
experimental groups for touchdown and gradient PCR, with only the DNA ladder bands
visible, implying unsuccessful amplification in all 20 tubes of four groups. This highly
suggests that several factors other than personal error contributed to the failure of the
experiment.

Several potential causes of error can be attributed to the absolute lack of amplification.
Primarily, the concentration and quality of the genomic DNA could have been too low for
successful amplification. Low DNA concentrations below threshold values or degraded
samples result in failed PCR amplification. The presence of contaminants such as proteins
inhibits PCR, therefore, a high-quality sample could be used instead [1]. Second, pipetting
errors might have played a role in the absence of amplification, smaller volumes have higher
error chances, especially with the usage of poorly calibrated or malfunctioning pipettes. Even
slight pipetting errors can significantly impact the PCR reaction [2].

Another presumed possible concern is the quality of the reagents, such as Taq polymerase,
primers, and MgCl2. In poor storage conditions, they could have lost activity, leading to
failed amplification. Taq polymerase is highly sensitive, and improper storage affects its
efficiency and amplification ability [3]. Although Gradient PCR was used to test a range of
multiple temperatures, incorrect gradient range settings or a narrow range might have led to
poor annealing conditions [4].

In conclusion, the experiment did not provide the desired results. It is crucial to check the
quality and concentration of DNA before starting the PCR reaction. Running a positive
control and a negative control along with the samples will help identify whether the PCR is
the problem or not. Moreover, optimizing the annealing temperatures, and ensuring accurate
pipetting and the usage of well calibrated pipettes can help resolve issues of non-specific
amplification. Finally, verifying the quality of reagents will be an important step in achieving
successful amplification. Repeating the experiment with the listed improvements will result
in better amplifications.

7. References:

[1] Haliç University Faculty of Science and Literature, Department of Molecular Biology and
Genetics. (2024). Molecular Biology Applications Laboratory-I Manual: Experiment 8 -
Polymerase Chain Reaction (PCR) optimization using touchdown PCR.

[2] Mullis, K., & Faloona, F. (1987). Specific synthesis of DNA in vitro via a polymerase-
catalyzed chain reaction. Methods in Enzymology, 155, 335–350.

[3] Dieffenbach, C. W., & Dveksler, G. S. (1995). PCR Primer: A Laboratory Manual. Cold
Spring Harbor Laboratory Press.
Roux, K. H. (1995). Optimization and troubleshooting in PCR. Cold Spring Harbor
Protocols, 1995(1), pdb.prot095108.

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[4] Korbie, D. J., & Mattick, J. S. (2008). Touchdown PCR for increased specificity and
sensitivity in PCR amplification. Nature Protocols, 3(9), 1452–1456.

[5] McPherson, M. J., & Møller, S. G. (2000). PCR: Basics from Background to Bench.
Springer Science & Business Media.

[6] Dieffenbach, C. W., Lowe, T. M., & Dveksler, G. S. (1993). General concepts for PCR
primer design. PCR Methods and Applications, 3(3), S30–S37.

[7] Innis, M. A., & Gelfand, D. H. (1990). Optimization of PCRs. In PCR Protocols: A Guide
to Methods and Applications. Academic Press.

[8] https://www.youtube.com/watch?v=_dpOT4dBp2g Access date: 12/12/2024