PCR: Polymerase Chain

Reaction - Group 7
Presented by:
Ananya - IIT2021061
Jyothsna Niharika - IIT2021055
Jay Mandal - IIT2021053
Contributions
1. Jyothsna Niharika
❖ Introduction to PCR
❖ Discovery of PCR
❖ PCR Components

1. Ananya
❖ PCR Procedure
❖ Types of PCR
❖ Example of PCR

1. Jay Mandal
❖ PCR Principle
❖ Advantages and Disadvantages of PCR
❖ Applications of PCR
❖ Conclusion
Table of contents
01. 04. 07.
Introduction to PCR PCR Procedure Adv and Disadv of PCR

02. 05. 08.

Discovery of PCR Types of PCR Applications of PCR

03. 06. 09.

Discovery of PCR Example of PCR Conclusion
01.
Introduction to PCR
What is PCR?
1. PCR or Polymerase Chain Reaction is a technique used in molecular biology to create several
copies of a certain DNA segment.

2. This tool is commonly used in the molecular biology and biotechnology labs. It is a laboratory
version of DNA Replication in cell which is commonly called “in vitro” since it occurs in a test
tube while “in vivo” signifies occurring in a living cell.

3. DNA Polymerase synthesises new strands of DNA complementary to the template DNA. The
DNA polymerase can add a nucleotide to the pre-existing 3’-OH group only. Therefore, a
primer is required. Thus, more nucleotides are added to the 3’ prime end of the DNA
polymerase.
Amplification of a specific target sequence

1. PCR does not copy all of the DNA in the sample. It copies only a very
specific sequence of genetic code from a template DNA, targeted by
PCR primers.

2. From this information two synthetic oligonucleotide primers may be

chemically synthesised each complementary to a stretch of DNA to the
3’ side of the target DNA, one oligonucleotide for each of the two DNA
strands (DNA polymerase can add a nucleotide only onto a preexisting
3'-OH group).
Why 2 primers?
1. In a PCR reaction you need two primers to amplify the target sequence one
called: Forward primer, which have the same sequence of forward DNA
strand and bind to the complementary reverse strand and the second called:
Reverse primer, which have the same sequence of reverse DNA strand and
bind to the complementary forward strand.

2. If there is only one primer, only one strand of the double stranded DNA will
be amplified in the PCR reaction.
 02.
Discovery of PCR
1. In the mid-1980s, Kary Mullis had a groundbreaking idea during a late-night drive along the
California coast: to amplify DNA in a test tube.

2. Working at Cetus Corporation, he developed the Polymerase Chain Reaction (PCR) technique, which
revolutionized genetic research and diagnostics.

3. By exploiting DNA replication processes through repeated cycles of temperature changes, Mullis
successfully demonstrated the amplification of specific DNA sequences in 1985.

4. This achievement earned him the Nobel Prize in Chemistry in 1993. PCR's continuous refinement
made it an indispensable tool in laboratories worldwide, driving advancements across various
scientific disciplines.

5. Today, PCR stands as a testament to human ingenuity, facilitating our understanding of the natural
world and enabling diverse applications of DNA technology.
 03.
Components of PCR
Principle: To amplify a segment of DNA using PCR, the sample is first heated so the DNA denatures,
or separates into two pieces of single-stranded DNA. Next, an enzyme called "Taq polymerase"
synthesizes - builds - two new strands of DNA, using the original strands as templates. This process
results in the duplication of the original DNA, with each of the new molecules containing one old and
one new strand of DNA. Then each of these strands can be used to create two new copies, and so on, and
so on .

Component Description

The sample DNA containing the target sequence. High temperature is applied to separate the strands at
DNA template
the beginning of the reaction.

DNA Polymerase Enzyme responsible for synthesizing new DNA strands by adding nucleotides to primer sequences. Taq
polymerase is commonly used due to its heat resistance.

Short single-stranded DNA sequences (~20 nucleotides) that bind to the template DNA and provide a
DNA Primers
starting point for DNA synthesis. Two primers (forward and reverse) are used.
Component Description

Essential cofactors for DNA polymerase activity. Magnesium ions (Mg^2+) are commonly
Cations
used, while manganese ions (Mn^2+) can increase error rates for DNA mutagenesis.

Thermal Cycler Instrument used to heat and cool reaction tubes to achieve specific temperatures required at
each step of the PCR reaction.

Deoxynucleoside Triphosphates are building blocks of DNA synthesis, supplying nucleotide

substrates for DNA polymerase to elongate strands during PCR.
(dNTPs)

Buffer Solution Provides optimal chemical environment for DNA polymerase activity and stability during PCR,
maintaining pH and ionic conditions necessary for efficient DNA amplification.
 04.
PCR Procedure
In PCR, nucleic acid is first extracted (released) from the organism or a clinical sample potentially
containing the target organism by heat, chemical, or enzymatic methods. Once extracted, target nucleic
acid is added to the reaction mix containing all the necessary components for PCR (primers, nucleotides,
covalent ions, buffer, and enzyme) and placed into a thermal cycler to undergo amplification which is
governed by temperature in following steps.

1. Denaturation (94-95°C)
● The reaction mixture is heated to 94-95 ⁰C, for between 15 and 30 seconds.
● The high temperature causes the hydrogen bonds between the bases in two strands of template
DNA to break and the two strands to separate.This results in two single strands of DNA, which
will act as templates to produce the new copies of each strand of DNA.
● It is important that the temperature is maintained at this stage for long enough to ensure that the
DNA strands have separated completely.
2. Annealing (55-65°C)
● The reaction is cooled to enable the primers to attach to a specific location on the single-stranded
template DNA by way of hydrogen bonding.
● The temperature depends on the characteristics of the primer, but is usually between 50 and 65 ⁰C.
● The two separated strands of DNA are complementary and run in opposite directions (from one
end – the 5’ end – to the other – the 3’ end). As a result, there are two primers – a forward primer
and a reverse primer.
● This step is essential because the primers serve as the starting point for DNA synthesis, by
providing a short region of double stranded DNA for the polymerase enzyme to work with. Only
once the primer has bound can the polymerase enzyme attach and start making the new
complementary strand of DNA from the loose DNA bases, in the extending step.
● The annealing step usually takes about 10-30 seconds.
3. Elongation (72°C)
● The heat is increased to 72 ⁰C to enable the new DNA to be made by a special Taq DNA polymerase
enzyme which adds DNA bases.
● Taq DNA polymerase is an enzyme taken from the bacteria Thermus aquaticus (“Taq”):
❖ This bacterium normally lives in hot springs so can tolerate temperatures above 80 ⁰C, but its
optimum temperature is 72⁰C.
❖ The bacteria’s DNA polymerase is very stable at high temperatures, which means it can withstand the
temperatures needed to break the strands of DNA apart in the denaturing stage of PCR.
● At 72⁰C, the Taq polymerase begins to build the complementary strand. It attaches to the primer
and then adds DNA bases to the single strand one-by-one in the 5’ to 3’ direction.
● The result is a brand-new strand of DNA and a double-stranded molecule of DNA.
● The duration of this step depends on the length of DNA sequence being amplified. It usually takes
around one minute to copy 1,000 DNA bases.
4. Repeating the process
● These three processes of thermal cycling are repeated 20-40 times to produce lots of copies of the DNA
sequence of interest.
● The new fragments of DNA that are made during PCR also serve as templates to which the DNA
polymerase enzyme can attach and start making DNA.
5. Using gel electrophoresis to visualize the results of PCR

● The results of a PCR reaction are usually visualized (made

visible) using gel electrophoresis. Gel electrophoresis is a
technique in which fragments of DNA are pulled through a gel
matrix by an electric current, and it separates DNA fragments
according to size.

● A standard, or DNA ladder, is typically included so that the size

of the fragments in the PCR sample can be determined.

● DNA fragments of the same length form a "band" on the gel,

which can be seen by eye if the gel is stained with a DNA-
binding dye
 05.
Types of PCR
Hot Start PCR Modified DNA polymerases remain inactive at lower temperatures, preventing nonspecific
amplification. Activation occurs at optimal temperature, enhancing specificity.

RNA is reverse transcribed into cDNA using reverse transcriptase, then amplified using PCR.
RT-PCR
Crucial for gene expression analysis and detecting RNA viruses like HIV.

Involves two successive PCR reactions with inner primers targeting the product of the first
Nested PCR PCR. Increases specificity and sensitivity, useful for amplifying diluted or degraded DNA
samples.

Simultaneously amplifies multiple target sequences within a single reaction using multiple
Multiplex PCR primer sets. Saves time, reagents, and sample material, commonly used in genotyping and
pathogen detection.

Also known as real-time PCR, quantifies DNA or RNA using fluorescent dyes or probes.
Quantitative
Highly sensitive and accurate, used for gene expression analysis, viral load measurement, and
PCR
detecting genetic variations.
06.
Example of PCR
1. We want to study a mutation in a DLG3 gene and how it relate to memory

2. Determine your target region

3. Design the primers using primer design tool, eg.Primer3

4. The region to be studied should be between the forward and reverse primer

5. Start PCR
1. Denaturation (95°C)
2. Annealing (58°C)
3. Extension (72°C)
Cycle #1 Cycle #2
07.
Advantages and
Disadvantages of PCR
Detects and amplifies small Rapid amplification within hours
DNA amounts

Adaptable to genotyping, sequencing,

Targeted amplification with ADVANTAGE
S cloning, mutation analysis, gene
specific primers
expression profiling

Easily automated for High sensitivity for

enhanced throughput detecting minute DNA
quantities
 Sensitivity to contamination Preferential amplification of
certain DNA sequences

Requirement for specialized Limited capacity for amplifying long

DISADVANT
equipment, reagents, and AGES DNA fragments
expertise

Introduction of Dependence on thermal

errors during cycling equipment
amplification
08.
Applications of PCR
Conclusion
In summary, Polymerase Chain Reaction (PCR) is a versatile and powerful technique widely used
across numerous fields. Its ability to amplify specific DNA sequences rapidly and accurately has
revolutionized genetic testing, forensic analysis, and medical diagnostics. In addition to its
applications in research and medicine, PCR plays a crucial role in environmental science by aiding
in biodiversity monitoring and bioremediation efforts. Furthermore, PCR is instrumental in
infectious disease control, enabling rapid pathogen detection and tracking of disease outbreaks.
References
1. Byju's: https://byjus.com/biology/pcr/
2. NCBI: https://www.ncbi.nlm.nih.gov/probe/docs/techpcr/
3. Genome:
https://www.genome.gov/about-genomics/fact-sheets/Polymerase-Chain-Reaction-Fact-Sheet
4. YourGenome: https://www.yourgenome.org/theme/what-is-pcr-polymerase-chain-reaction/
5. Addgene: https://www.addgene.org/protocols/pcr/
6. Genome:
https://www.genome.gov/genetics-glossary/Polymerase-Chain-Reaction#:~:text=Polymerase c
hain reaction
7. KSU: https://faculty.ksu.edu.sa/sites/default/files/ppt.polymerase_chain_reaction_pcr.pdf