Explain how Polymerase Chain Reaction (PCR) works and outline its fundamentalprocedural steps.
Polymerase Chain Reaction (PCR)
PCR is an in-vitro technique used to target and amplify a specific region of a DNA strand
to generate large quantities of a specified DNA from a small quantity.
• History: Kary Mullis conceived the idea in 1983, introduced it to the scientific
community in 1985, and was awarded the Nobel Prize for this discovery in 1987.
• Principle: Double-stranded DNA is converted to single-stranded DNA using heat.
Two oligonucleotide primers complementary to the 3’ end of each DNA strand are
used. These primers attach to the 3’ end, and Taq polymerase extends the DNA by
incorporating nucleotides.
• Stages:
1. Exponential Amplification: The amount of product doubles with every cycle.
The reaction is very sensitive.
2. Levelling of Stage: The reaction slows down as DNA polymerase activity
decreases.
3. Plateau: No more product accumulates due to exhaustion of reagents and
enzymes.
,• Requirements:
o Template DNA: A segment of DNA to be amplified, extracted from the
sample.
o Reaction buffer: Provides ionic strength and buffering capacity (e.g., Tris-
HCl, ammonium ions, KCl).
o Monovalant and Divalant cations: Such as Magnesium Chloride and
Potassium, which work as a co-factor for the enzyme.
o Primers: Small, artificially made DNA strands, typically 20-30 nucleotides
long. They are complementary to the 3’ end of the target DNA and include a
forward primer and a reverse primer. An oligo primer is generally 18–24
bases long.
o DNA polymerase: Attaches at the end of the primer and adds new
nucleotides to the DNA strand at the 3′ end complementary to the target
DNA. Taq Polymerase, extracted from Thermus aquaticus, is used because
it works efficiently at higher temperatures.
o Deoxynucleotide Triphosphates (dNTPs): dATP, dCTP, dGTP, and dTTP,
serving as the basic building blocks for new DNA strands.
o PCR Machine: A thermal cycler.
• Steps:
1. Denaturation: At 94-98°C for 1 to 2 minutes, double-stranded DNA melts
and opens into two single strands.
2. Annealing: At medium temperatures (around 45-60°C), primers pair up
(anneal) with the single-stranded template. Polymerase attaches to the short
double-stranded region.
3. Extension: At 72°C (the optimal temperature for Taq polymerase), the
polymerase adds complementary nucleotides from the 3’ end to the 5’ end of
the DNA.
, • At least 30 cycles of these three steps are typically performed. This leads to an
exponential increase in the number of genes in each cycle.
• Analysis of PCR Product: Can be done using Agarose gel electrophoresis (to
check for bands, their size, presence of a smear, or a single sharp band), Cloning of
Product (when the gene is in a tiny amount), or Sequencing of Product (using an
automated sequencer).
• Applications of PCR (listed under Oligo Primers but apply to PCR): Amplifying DNA,
RT-PCR, DNA Sequencing, Gene Cloning, Mutation Detection.
• Primer Design: An oligo primer is defined as a synthetic strand of nucleotides,
typically 18–24 bases long. Some sources state they are 20-30 nucleotides. Primers
are small pieces of artificially made DNA strands. They are designed to be
complementary to the 3’ end of each strand of the target DNA. In a PCR, primers
attach or pair up (anneal) with the single-stranded template DNA at medium
temperatures, around 45-60°C. This binding allows the DNA polymerase to start
copying the template. Two primers are used in PCR: a forward primer and a reverse
primer. The forward primer binds to the start of the sense strand, while the reverse
primer binds to the start of the antisense strand. Both primers are necessary in PCR
to amplify a specific DNA region.
• Primer design is a crucial step for various molecular techniques such as PCR, RT-
PCR, DNA sequencing, gene cloning, and mutation detection. For cloning by PCR,
primers specific to the gene of interest are designed, often with added restriction
Polymerase Chain Reaction (PCR)
PCR is an in-vitro technique used to target and amplify a specific region of a DNA strand
to generate large quantities of a specified DNA from a small quantity.
• History: Kary Mullis conceived the idea in 1983, introduced it to the scientific
community in 1985, and was awarded the Nobel Prize for this discovery in 1987.
• Principle: Double-stranded DNA is converted to single-stranded DNA using heat.
Two oligonucleotide primers complementary to the 3’ end of each DNA strand are
used. These primers attach to the 3’ end, and Taq polymerase extends the DNA by
incorporating nucleotides.
• Stages:
1. Exponential Amplification: The amount of product doubles with every cycle.
The reaction is very sensitive.
2. Levelling of Stage: The reaction slows down as DNA polymerase activity
decreases.
3. Plateau: No more product accumulates due to exhaustion of reagents and
enzymes.
,• Requirements:
o Template DNA: A segment of DNA to be amplified, extracted from the
sample.
o Reaction buffer: Provides ionic strength and buffering capacity (e.g., Tris-
HCl, ammonium ions, KCl).
o Monovalant and Divalant cations: Such as Magnesium Chloride and
Potassium, which work as a co-factor for the enzyme.
o Primers: Small, artificially made DNA strands, typically 20-30 nucleotides
long. They are complementary to the 3’ end of the target DNA and include a
forward primer and a reverse primer. An oligo primer is generally 18–24
bases long.
o DNA polymerase: Attaches at the end of the primer and adds new
nucleotides to the DNA strand at the 3′ end complementary to the target
DNA. Taq Polymerase, extracted from Thermus aquaticus, is used because
it works efficiently at higher temperatures.
o Deoxynucleotide Triphosphates (dNTPs): dATP, dCTP, dGTP, and dTTP,
serving as the basic building blocks for new DNA strands.
o PCR Machine: A thermal cycler.
• Steps:
1. Denaturation: At 94-98°C for 1 to 2 minutes, double-stranded DNA melts
and opens into two single strands.
2. Annealing: At medium temperatures (around 45-60°C), primers pair up
(anneal) with the single-stranded template. Polymerase attaches to the short
double-stranded region.
3. Extension: At 72°C (the optimal temperature for Taq polymerase), the
polymerase adds complementary nucleotides from the 3’ end to the 5’ end of
the DNA.
, • At least 30 cycles of these three steps are typically performed. This leads to an
exponential increase in the number of genes in each cycle.
• Analysis of PCR Product: Can be done using Agarose gel electrophoresis (to
check for bands, their size, presence of a smear, or a single sharp band), Cloning of
Product (when the gene is in a tiny amount), or Sequencing of Product (using an
automated sequencer).
• Applications of PCR (listed under Oligo Primers but apply to PCR): Amplifying DNA,
RT-PCR, DNA Sequencing, Gene Cloning, Mutation Detection.
• Primer Design: An oligo primer is defined as a synthetic strand of nucleotides,
typically 18–24 bases long. Some sources state they are 20-30 nucleotides. Primers
are small pieces of artificially made DNA strands. They are designed to be
complementary to the 3’ end of each strand of the target DNA. In a PCR, primers
attach or pair up (anneal) with the single-stranded template DNA at medium
temperatures, around 45-60°C. This binding allows the DNA polymerase to start
copying the template. Two primers are used in PCR: a forward primer and a reverse
primer. The forward primer binds to the start of the sense strand, while the reverse
primer binds to the start of the antisense strand. Both primers are necessary in PCR
to amplify a specific DNA region.
• Primer design is a crucial step for various molecular techniques such as PCR, RT-
PCR, DNA sequencing, gene cloning, and mutation detection. For cloning by PCR,
primers specific to the gene of interest are designed, often with added restriction
