Name of the author: Çağatay Utku ÇAMYAR

Date of the experiment: 28.12.2023

Submission date: 04.01.2024
Name of the experiment: PCR Technique and Primer Design for Mutation Detection
Evaluator: Fatma AYDINOĞLU

PCR TECHNIQUE and PRIMER DESIGN for MUTATION DETECTION

Çağatay Utku ÇAMYAR, Tuba KENAR, Hatice ÇELİK, İrem OĞUZHAN, Deniz DURMAZ,
Hümeyra KELEŞ, İbrahim YAĞŞİ
ABSTRACT

The PCR (Polymerase Chain Reaction) technique is a powerful molecular biology tool
that enables the targeted amplification of specific DNA sequences. In this experiment, we aimed
to learn the principles and usage areas of PCR. This experiment involved three main steps:
denaturation, annealing, and extension. Understanding these three main steps is very important
to achieve our aim for this experiment. The success of the PCR reaction was assessed by
analysing the amplified products using agarose gel electrophoresis. The results confirmed the
successful amplification of the target DNA fragment, highlighting the efficiency and specificity
of the PCR technique.

Primer design is a critical step in the PCR (Polymerase Chain Reaction) process,
influencing the specificity and efficiency of DNA amplification. This laboratory experiment
also aimed to practice primer design and to learn diagnosing genetic diseases by genetic
screening. In the end of this experiment, we observe that the results of this experiment
demonstrated successful amplification of the target gene, validating the importance of
thoughtful primer design for robust PCR assays.

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INTRODUCTION

In this experiment we investigate and learn about what is PCR and how its use. To do
this we prepare a master mix and took some of them to use in our experiment. Also, we used
template DNA for our observation in this experiment. In the end, the result we obtained
observed and discussed.

PCR (Polymerase Chain Reaction) is a widely utilized technique in molecular biology

for the amplification of specific DNA sequences. Developed by Kary Mullis in the 1980s, PCR
has revolutionized various fields, including genetics, forensics, and medical diagnostics [1]. In
this lab report we aimed to learn the principles and usage areas of PCR which is the basic
techniques used in molecular biology laboratories. The method involves a cyclic process that
replicates a DNA template exponentially, producing millions of copies of a target sequence.

In genetic research, PCR facilitates the amplification of specific DNA sequences, aiding
in gene expression studies and the identification of disease-related mutations [2]. In medical
diagnostics, PCR has become indispensable for detecting infectious agents, such as viruses and
bacteria, contributing significantly to disease diagnosis [3]. Forensic science relies on PCR for
DNA profiling, aiding criminal investigations and paternity testing and pharmacogenomics
utilizes PCR to identify genetic variations influencing drug responses, paving the way for
personalized medicine [4]. Environmental studies leverage PCR to analyse microbial diversity
in ecosystems, contributing to our understanding of environmental health. In evolutionary
biology, PCR assists in tracing genetic relationships among species [5]. This illustrates the
versatility of PCR, highlighting its central role in advancing molecular biology across diverse
scientific domains. The PCR is not naturally present in every cell. It is a method used to amplify
specific DNA sequences in a controlled environment, typically in a laboratory setting.

Also, we used taq polymerase in this experiment. Taq polymerase is a heat-stable DNA
polymerase enzyme derived from the bacterium Thermus aquaticus. It is a crucial component
in the Polymerase Chain Reaction (PCR) process. Taq polymerase is known for its ability to
withstand the high temperatures (around 94–98°C) required for DNA denaturation during each
cycle of PCR. Taq polymerase has played a pivotal role in making PCR a widely used and
efficient technique in molecular biology and genetics research.

The Rubisco gene encodes the ribulose-1,5-bisphosphate carboxylase/oxygenase

enzyme, a key player in photosynthesis [6]. Found in the chloroplasts of plants rubisco catalyses
the fixation of carbon dioxide during photosynthesis.

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MATERIALS AND METHODS

In this experiment, 6 µl F primer, 6 µl R primer, 15 µl taq polymerase, 15 µl enzyme

buffer, and 99 µl water used to prepare a master mix. Then 49 µl taken from that master mix
and added to an eppendorf tube with 1 µl of template DNA. The denaturation of DNA is the
reaction mixture, containing DNA template, primers, Taq polymerase, nucleotides, and buffer,
was heated to 94-98°C, causing DNA denaturation. 3 different method were used during the
experiment. First one called annealing and it is the reaction temperature was lowered to allow
primers to anneal to the complementary sequences on the template DNA. Second one called
extension and it means that taq polymerase extended the primers by adding nucleotides to the
3' end, synthesizing a complementary strand of DNA. Last one called cycling and it is the steps
were repeated for 20-40 cycles to achieve exponential amplification.

An essential consideration in the design of the PCR assay lies in the length of the primer
harbouring the desired forced mutation. The replication of DNA commences at the 5' end of the
primer during annealing and intentional mismatch incorporation. Consequently, the PCR
product initiated from this process is limited by the length of the primer. It is imperative that
the primer utilized in the allele-specific positive control test does not surpass the length of the
site-directed mutagenesis primer. Striking a balance, however, is crucial, as excessively short
allele-specific primers, measuring below 18 base pairs, heighten the risk of non-specific binding
to non-target DNA. To circumvent the challenges associated with lengthy primers, strategic
placement of the forced mutation near the 3' end of the oligo is recommended, allowing
sufficient remaining length for accommodating the allele-specific primer.

RESULTS

The results of this experiment demonstrated successful amplification of the target DNA
sequence. We revealed bands corresponding to the expected sizes of the amplified fragments.
The presence of these bands confirmed the specificity of the PCR reaction, indicating that the
designed primers successfully targeted the desired regions within the genomic DNA. In the end
of the experiment, we observed some similar results with the other groups. Learned to generate
expected DNA products suitable for further applications such as sequencing or analysis. The
outcome supports the effectiveness of the chosen primer design and the overall reliability of the
PCR technique in selectively amplifying specific DNA sequences.

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DISCUSSION

This experiment involves several crucial steps for the selective amplification of specific
DNA sequences. Subsequently, during the annealing step, specific primers bind to
complementary sequences on the single-stranded DNA template. The extension step follows,
wherein a heat-stable DNA polymerase synthesizes a complementary strand by adding
nucleotides to the primers. This denaturation-annealing-extension cycle is repeated multiple
times, resulting in exponential amplification of the target DNA. A final extension step
completes the synthesis of partially extended strands. Each step is crucial, and the success of
the PCR relies on careful primer design, precise temperature control, and appropriate cycling
conditions to ensure specificity, efficiency, and accurate replication of the target DNA sequence.

In this experiment, each chemical and solution plays a crucial role in ensuring the
success of the DNA amplification process. The DNA template serves as the blueprint,
containing the target sequence for amplification. Primers are short, single-stranded DNA
sequences designed to bind to the complementary regions flanking the target sequence, serving
as initiation points for DNA synthesis. Taq polymerase, a heat-stable DNA polymerase, is the
enzymatic workhorse responsible for synthesizing new DNA strands during the extension step.
The nucleotide mix provides the necessary adenine, thymine, cytosine, and guanine nucleotides,
serving as the building blocks for the growing DNA strands. The buffer solution maintains the
optimal pH for Taq polymerase activity, ensuring efficient DNA amplification. The combination
of these components is essential for the specificity, efficiency, and accuracy of the PCR
technique.

Working with ice during PCR is essential to maintain the stability of reaction
components and prevent undesired interactions. The low temperature helps preserve the activity
of heat-sensitive enzymes, particularly DNA polymerases, ensuring their functionality during
subsequent temperature cycles. Additionally, ice minimizes the risk of nonspecific
amplification by slowing molecular movements. So, working on ice is a critical measure that
contributes to the reliability and success of PCR reactions.

In this experiment we might have problems like contamination, where unwanted

substances can give incorrect results. Issues with the primers, which are tiny bits of DNA,
causing the experiment to fail or show the wrong information. The DNA sample quality might
not be good enough, affecting the success of the experiment. These are some possible errors
that we can do during the experiment.

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REFERENCES

[1] Mullis, K. B. (1994). The polymerase chain reaction (Vol. 41, No. 5). Springer science &
business media.

[2] Mullis, K. B. (1990). The unusual origin of the polymerase chain reaction. Scientific
American, 262(4), 56-65.

[3] Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., ... & Erlich,
H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA
polymerase. Science, 239(4839), 487-491.

[4] Evans, W. E., & Relling, M. V. (1999). Pharmacogenomics: translating functional genomics
into rational therapeutics. science, 286(5439), 487-491.

[5] Hillis, D. M., & Dixon, M. T. (1991). Ribosomal DNA: molecular evolution and
phylogenetic inference. The Quarterly review of biology, 66(4), 411-453.

[6] Whitney, S. M., & Andrews, T. J. (2001). Plastome-encoded bacterial ribulose-1, 5-

bisphosphate carboxylase/oxygenase (RubisCO) supports photosynthesis and growth in
tobacco. Proceedings of the National Academy of Sciences, 98(25), 14738-14743.