Biology PPT - Polymerase Chain Reaction

PCR: Polymerase
Chain Reaction
Presented by:
Ananya - IIT2021061
Jyothsna Niharika - IIT2021055
Jay Mandal - IIT2021053
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
DNA template The sample DNA containing the target sequence. High temperature is applied to separate
the strands at 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
DNA Primers
provide a 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
Cations commonly 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)
Provides optimal chemical environment for DNA polymerase activity and stability
Buffer Solution
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 (95 degree)

● The reaction mixture is heated to 94-95⁰C, 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 degree)
● 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 65⁰C.
● 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 degree)
● The heat is increased to 72⁰C 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 80⁰C, 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
Modified DNA polymerases remain inactive at lower temperatures, preventing
Hot Start PCR
nonspecific amplification. Activation occurs at optimal temperature, enhancing
specificity.
RNA is reverse transcribed into cDNA using reverse transcriptase, then amplified
RT-PCR
using PCR. Crucial for gene expression analysis and detecting RNA viruses like HIV.
Involves two successive PCR reactions with inner primers targeting the product of
Nested PCR the first PCR. Increases specificity and sensitivity, useful for amplifying diluted or
degraded DNA samples.
Simultaneously amplifies multiple target sequences within a single reaction using

Multiplex PCR multiple 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
Quantitative
probes. Highly sensitive and accurate, used for gene expression analysis, viral load
PCR
measurement, and 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 Rapid amplification within
small DNA amounts hours
Adaptable to genotyping,
Targeted amplification
ADVANTAGES sequencing, cloning, mutation
with specific primers
analysis, gene expression
profiling
Easily automated High sensitivity for

for enhanced detecting minute DNA
throughput quantities
Sensitivity to Preferential amplification of
contamination certain DNA sequences
Requirement for Limited capacity for amplifying

DISADVANT long DNA fragments
specialized equipment, AGES
reagents, and 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.

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PCR: Polymerase

02. 05. 08.

03. 06. 09.

3. DNA Polymerase synthesises new strands of DNA complementary to the template

2. From this information two synthetic oligonucleotide primers

2. Working at Cetus Corporation, he developed the Polymerase Chain Reaction (PCR)

3. By exploiting DNA replication processes through repeated cycles of temperature changes,

5. Today, PCR stands as a testament to human ingenuity, facilitating our understanding of

Deoxynucleoside Triphosphates are building blocks of DNA synthesis, supplying

1. Denaturation (95 degree)

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

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

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

Simultaneously ampliﬁes multiple target sequences within a single reaction using

2. Determine your target region

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

Easily automated High sensitivity for

Requirement for Limited capacity for amplifying

Introduction of Dependence on thermal

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