PCR and Gel Electrophoresis

The polymerase chain reaction (PCR) and gel electrophoresis are two of the most important techniques in modern molecular biology. PCR allows the amplification of a specific DNA sequence from a tiny sample, while gel electrophoresis separates DNA fragments by size. Together, they are used in forensics, medical diagnosis, research, and genetic engineering. Both techniques are specified in the AQA A-Level Biology syllabus.

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The Polymerase Chain Reaction (PCR)

Key Definition: PCR is an in vitro technique that rapidly amplifies (makes millions of copies of) a specific DNA sequence from a very small starting sample.

PCR was developed by Kary Mullis in 1983 and has revolutionised molecular biology. It works by mimicking DNA replication in a test tube using a thermal cycler.

Components Required for PCR

Component	Role
Template DNA	The DNA sample containing the target sequence to be amplified
Primers	Short (typically 18–25 bases), single-stranded DNA sequences that are complementary to sequences flanking the target region. Two primers are needed — one for each strand (forward and reverse primers)
Taq DNA polymerase	A heat-stable DNA polymerase isolated from Thermus aquaticus, a thermophilic bacterium found in hot springs. It can withstand repeated cycles of high temperature without being denatured
Free deoxyribonucleotides (dNTPs)	The four DNA nucleotides (dATP, dTTP, dCTP, dGTP) used as building blocks for the new DNA strands
Buffer solution	Provides optimal pH and ion concentrations (including Mg²⁺, which is a cofactor for Taq polymerase)

The Three Steps of a PCR Cycle

Each PCR cycle has three temperature-dependent steps:

1. Denaturation (94–98°C, typically ~30 seconds)

• The high temperature breaks the hydrogen bonds between complementary base pairs.
• The double-stranded DNA separates into two single strands.
• This provides single-stranded templates for primer binding.

2. Annealing (50–65°C, typically ~30 seconds)

• The temperature is lowered to allow the primers to bind (anneal) to their complementary sequences on the template DNA.
• The exact annealing temperature depends on the length and base composition of the primers (higher GC content = higher annealing temperature, because G≡C has three hydrogen bonds vs. A=T with two).
• Primers bind before the original DNA strands can re-anneal because primers are present in vast excess and are shorter (so they bind more quickly).

3. Extension (72°C, typically 1 minute per kb of target)

• Taq polymerase synthesises new DNA strands by adding complementary nucleotides to the 3' end of each primer.
• Extension occurs in the 5' to 3' direction (as with all DNA polymerases).
• 72°C is the optimal temperature for Taq polymerase activity.

Exponential Amplification

• Each cycle doubles the number of target DNA molecules.
• After n cycles, the number of copies = 2ⁿ (assuming 100% efficiency).
• A typical PCR run involves 25–35 cycles.
• After 30 cycles, a single molecule of DNA would be amplified to approximately 2³⁰ = 1.07 billion copies.

Worked Example — PCR Amplification:

A forensic scientist begins with 10 copies of a target DNA sequence. How many copies will be present after 20 cycles of PCR?

Solution: Number of copies = 10 × 2²⁰ = 10 × 1,048,576 = 10,485,760 copies (approximately 10.5 million).

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Why Use Taq Polymerase?

• Conventional DNA polymerases (e.g., from E. coli) would be denatured at 94–98°C and would need to be replenished after each cycle.
• Taq polymerase is thermostable — it remains active at high temperatures because it evolved in a thermophilic bacterium living at ~80°C.
• Its optimal activity temperature is ~72°C, which provides good specificity and extension rate.

Applications of PCR

• Forensic science: Amplifying DNA from crime scenes (blood, saliva, hair) for genetic fingerprinting.
• Medical diagnosis: Detecting pathogen DNA (e.g., COVID-19 RT-PCR tests), identifying genetic mutations.
• Paternity testing: Amplifying STR loci for comparison.
• Archaeology and palaeontology: Amplifying ancient DNA from fossils or preserved specimens.
• Research: Cloning genes, site-directed mutagenesis, sequencing preparation.

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Gel Electrophoresis

Key Definition: Gel electrophoresis is a technique that separates DNA (or RNA or protein) fragments according to their size (and charge) by passing them through a gel matrix under the influence of an electric field.