A common but revolutionary molecular biology technique developed by Kary B. Mullis in 1983, who later won the Nobel Prize in Chemistry for this invention. It is based on the natural process of DNA replication but is carried out in vitro (outside the living cell) using cyclic temperature changes to amplify a specific DNA region.
PCR selectively amplifies a target DNA sequence through repeated cycles of denaturation, annealing, and extension, using a thermostable DNA polymerase.
Each cycle doubles the number of target DNA copies, leading to exponential amplification, which can be mathematically represented:
Formula:
Number of DNA copies = N₀ × 2ⁿ
Where:
N₀ = Initial number of DNA copies (starting template)
n = Number of PCR cycles
2ⁿ = Amplification factor (since DNA doubles every cycle, theoretically)
Example:
If you start with 1 copy of DNA and run the PCR for 30 cycles: DNA copies = 1 × 2³⁰ = 1,073,741,824 copies That’s over 1 billion copies from just one molecule!
However, in reality, amplification is not perfectly exponential due to limiting reagents, enzyme efficiency, and accumulation of inhibitors. After ~30–35 cycles, the reaction plateaus because:
dNTPs get used up
Polymerase loses activity
Product re-annealing competes with primer binding
So while the formula gives a theoretical maximum, the actual yield is often lower.
Components Used in PCR Reaction
A PCR reaction requires a carefully balanced mix of chemical components, each serving a specific purpose. Below is a detailed breakdown of these components and their roles:
1. Template DNA
The starting material containing the target sequence to be amplified. It can be genomic DNA, cDNA, plasmid DNA, or even RNA (in reverse transcription PCR). The typical amount ranges from 1 ng to 1 µg, depending on the source and purity of the material. Too much or too little template can affect amplification efficiency.
2. Primers
Short, single-stranded DNA sequences (15–30 nucleotides) that are complementary to the flanking regions of the target DNA sequence. They provide a starting point for DNA polymerase to begin synthesis. Primers are designed to be specific to the target sequence, with minimal self-complementarity to avoid primer-dimer formation.
Want to know how a primer pair is designed? Read this article:
DNA polymerase synthesizes new DNA strands by adding deoxynucleotide triphosphates (dNTPs) to the growing chain.
Taq polymerase, derived from Thermus aquaticus, is the most commonly used enzyme due to its thermostability at high temperatures.
Other polymerases, like Pfu (higher fidelity) or Vent, are used for applications requiring greater accuracy.
The enzyme’s activity is optimal at 72°C and requires magnesium ions as cofactors.
Taq polymerase lacks 3′ to 5′ exonuclease proofreading activity, which means it cannot correct mistakes made during DNA synthesis. As a result, it tends to introduce errors (wrong nucleotides) more frequently. In contrast, high-fidelity enzymes like Pfu possess proofreading capability, enabling them to detect and correct mismatched bases during extension, making them more accurate for applications like cloning or sequencing.
4. Deoxynucleotide Triphosphates (dNTPs)
dNTPs (dATP, dCTP, dGTP, and dTTP) are the building blocks of the new DNA strands. They are typically added at a concentration of 50–200 µM each.
5. Buffer Solution
The buffer maintains the optimal pH and ionic environment for DNA polymerase activity. A standard 10× PCR buffer usually contains:
Tris-HCl (pH 8.3–8.8)
Potassium chloride (KCl)
Ammonium sulphate (in some protocols)
The buffer stabilizes the enzyme and ensures proper denaturation and annealing conditions.
6. Magnesium Chloride (MgCl₂)
Magnesium ions are essential cofactors for DNA polymerase, stabilizing the interaction between the enzyme, the template DNA, and dNTPs.
Typical concentration: 1.5–2.5 mM
Too little Mg²⁺ → Poor enzyme activity
Too much Mg²⁺ → Non-specific amplification
The optimal concentration may vary depending on the template, primers, and polymerase used.
PCR – The Process
Initial Denaturation
Temperature: 94–98°C
Duration: 2–5 minutes
Purpose: Ensures complete separation of the two complementary strands of DNA, especially important for genomic DNA.
Denaturation
Temperature: 94–98°C
Duration: 20–30 seconds (can go up to 60 seconds for GC-rich DNA)
Function: Melts the double-stranded DNA to create single-stranded templates.
Annealing
Temperature: 50–65°C
Duration: 20–40 seconds
The exact temperature is usually 3–5°C below the melting temperature (Tm) of the primers.
Function: Allows primers to bind specifically to their complementary sequences on the single-stranded DNA template.
Extension
Temperature: 72°C
Duration: 30–60 seconds
Function: DNA polymerase synthesizes the new DNA strand by adding dNTPs to the primer-bound template.
The steps (Denaturation → Annealing → Extension) are repeated for 20–40 cycles, depending on the experiment. PCR is performed in a thermal cycler, a device that precisely controls the rapid temperature changes needed for each step.
Thermal Cycler
Final Extension
Temperature: 68–72°C
Duration: 5–7 minutes
This step occurs after all cycles are completed and serves several important purposes:
Completes Any Partially Synthesized Strands
In the final cycle, some strands may not have been fully extended.
This step gives DNA polymerase time to finish any incomplete products.
Ensures Full-Length Amplicons
Especially important for longer PCR products or GC-rich regions, where synthesis can lag behind.
Enables A-overhang Addition (for Taq Polymerase)
Taq polymerase adds a single adenine (A) base at the 3’ end of the product, which is essential for TA cloning.
The final extension allows this to occur efficiently.
Compensates for Enzyme Degradation
Although Taq polymerase is thermostable, repeated heating during denaturation partially inactivates it over time.
Its activity decreases by the end of 30–40 cycles.
The final extension allows the remaining active enzyme to finish synthesis before it becomes completely inactive.
PCR Enhancers
PCR enhancers are additives used to increase efficiency, specificity, and yield, especially when typical conditions fail. They are particularly helpful for:
GC-rich templates
Long DNA fragments
Secondary structures
Low DNA concentration
Crude or inhibitor-rich samples
Common Enhancers
DMSO (Dimethyl Sulfoxide)
Weakens hydrogen bonds between DNA bases, lowering the melting temperature (Tm)
Breaks down secondary structures, increasing template accessibility
Betaine
Acts as both an osmoprotectant and an isostabilizing agent
For GC-rich regions: Lowers Tm by disrupting strong G–C bonding
For AT-rich regions: Increases Tm by reducing disorder (entropy) in single-stranded DNA
Overall effect: Makes different regions of DNA behave more uniformly
Glycerol
Stabilizes Taq polymerase, especially under tough conditions (e.g., high GC-content templates)
Helps maintain proper enzyme folding and activity throughout the reaction
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