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 — billions of copies from a single starting molecule, in a few hours, in a test tube. Gel electrophoresis separates DNA fragments by size, providing a way to verify and analyse the products of PCR, restriction digests, and DNA fingerprinting reactions. Together they underpin forensic science, medical diagnostics, paternity testing, conservation genetics, basic research, and genetic engineering. Both techniques are explicit AQA A-Level Biology content.

Spec mapping: This lesson sits in AQA 7402 Section 3.8.4 — Gene technologies allow the study and alteration of gene function. The specification expects candidates to describe the principle of PCR (including the role of primers, Taq DNA polymerase, free nucleotides, and the heating/cooling cycles), to calculate amplification yields, and to describe the principle and applications of gel electrophoresis. (Refer to the official AQA specification document for exact wording.)

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The Polymerase Chain Reaction (PCR)

Key Definition: PCR is an in vitro technique that rapidly amplifies (makes many millions of copies of) a specific DNA sequence from a very small starting sample. The reaction is performed in a single tube using a programmable heat block — the thermal cycler — that ramps the temperature up and down through the cycle phases.

PCR was developed by Kary Mullis in 1983 (Nobel Prize in Chemistry, 1993) and has revolutionised molecular biology. It works by mimicking DNA replication in a test tube — recreating, in successive temperature-controlled steps, the strand separation, primer binding and extension that ordinarily occur in vivo at a replication fork.

Components Required for PCR

Component	Role
Template DNA	The DNA sample containing the target sequence to be amplified. Quantities can be vanishingly small — a single cell, a fragment of bone, a smear of saliva
Primers	Short (typically 18–25 bases) single-stranded DNA sequences complementary to sequences flanking the target region. Two primers are needed — one for each strand. The forward primer anneals to the antisense strand and primes synthesis in the 5'→3' direction toward the gene; the reverse primer anneals to the sense strand and primes synthesis in the opposite direction
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⁺, an essential cofactor for Taq polymerase)

The Three Steps of a PCR Cycle

Each PCR cycle has three temperature-dependent steps. A typical reaction runs 25–35 cycles in approximately 1.5–3 hours.

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

• 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.
• The covalent (phosphodiester) backbone is not affected — only the secondary structure is disrupted.

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

• The temperature is lowered to allow the primers to anneal (bind) to their complementary sequences on the template DNA by complementary base pairing.
• The exact annealing temperature depends on the length and base composition of the primers — higher GC content allows higher annealing temperatures, because each G≡C pair has three hydrogen bonds compared with two for each A=T pair.
• Primers bind before the original DNA strands can fully re-anneal because primers are present in vast excess (~10⁹-fold more than template at the start of the reaction) and are shorter (so they encounter their targets faster).
• The 3' end of the primer points into the region to be amplified.

3. Extension (72°C, typically 30–60 s 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 on the new strand (as with all DNA polymerases — they cannot add nucleotides in the 3'→5' direction).
• 72°C is the optimal temperature for Taq polymerase activity. Lower temperatures slow extension; higher temperatures inactivate the enzyme.

Mermaid: one PCR cycle

flowchart TD
  A["Double-stranded template DNA + primers + dNTPs + Taq + Mg²⁺"] --> B["94-98°C Denaturation: H-bonds break, strands separate"]
  B --> C["50-65°C Annealing: primers H-bond to complementary template sequences"]
  C --> D["72°C Extension: Taq adds dNTPs at 3' end of primer, 5'→3'"]
  D --> E["Two double-stranded copies of region between primers"]
  E -.->|Cycle 2 onwards| B

Exponential Amplification

• Each cycle approximately 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.

This exponential growth is the conceptual core of PCR. After the first few cycles, the reaction is dominated by products that are exactly bounded by the two primers — fragments of defined length corresponding to the region between (and including) the primer binding sites. The original (longer) template strands are vastly outnumbered.

Worked Example 1 — 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 (assuming 100% efficiency)?

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

Worked Example 2 — Cycles required:

A diagnostic laboratory needs at least 10⁹ copies of a target sequence for reliable detection. Starting from a single molecule, how many PCR cycles are required (assuming 100% efficiency)?

Solution: Require 2ⁿ ≥ 10⁹. Take log₂: n ≥ log₂(10⁹) = 9 × log₂(10) ≈ 9 × 3.32 ≈ 29.9. So at least 30 cycles are required. (In practice, a margin is added — 35 cycles is typical.)

Why Use Taq Polymerase?

• Conventional DNA polymerases (e.g., the E. coli DNA polymerase I Klenow fragment) would be denatured at 94–98°C and would need to be replenished after each cycle. This was the case in early experiments before Taq was introduced, making PCR laborious and impractical.
• Taq polymerase is thermostable. It comes from Thermus aquaticus, a thermophilic bacterium isolated from hot springs in Yellowstone National Park. Its enzymes have evolved to operate at ~80°C and tolerate brief exposure to 95°C.
• Its optimal activity temperature is ~72°C, which provides good extension rate and specificity (high temperatures destabilise mismatched primer–template pairs).
• A practical consequence: Taq is added once at the start of the reaction. The thermal cycler then runs all cycles unattended.
• Higher-fidelity engineered thermostable polymerases (Pfu, Phusion) are used when the highest sequence accuracy is required (e.g. for cloning or sequencing); Taq has a relatively high error rate (~1 in 10⁵).

Variants of PCR

• Reverse-transcription PCR (RT-PCR) — starts from RNA. Reverse transcriptase first makes a cDNA copy; PCR then amplifies the cDNA. Used for measuring RNA expression and for diagnosing RNA viruses such as SARS-CoV-2.
• Quantitative PCR (qPCR) — fluorescent reporters allow real-time monitoring of amplification, making PCR quantitative rather than just qualitative.
• Multiplex PCR — multiple primer pairs in one reaction amplify several target sequences simultaneously. Used in STR-based DNA profiling (Lesson 6).

Applications of PCR

• Forensic science: amplifying DNA from crime scenes (blood, saliva, hair, skin cells) for genetic fingerprinting.
• Medical diagnosis: detecting pathogen DNA or RNA. The COVID-19 RT-PCR test was the global benchmark for SARS-CoV-2 detection during the pandemic. PCR also detects HIV, tuberculosis, hepatitis B and C, and many other infections.
• Paternity testing: amplifying STR loci for comparison between alleged parent and child.
• Archaeology and palaeontology: amplifying ancient DNA from fossils or preserved specimens. The Svante Pääbo lab's work on Neanderthal and Denisovan DNA — Nobel Prize 2022 — depended entirely on PCR (and modern descendants of it).
• 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 by passing them through a gel matrix under the influence of an electric field. For DNA the principal variable is size — smaller fragments migrate further; the charge per unit length is essentially constant for all DNA.

Principle

• DNA is negatively charged (due to the deprotonated phosphate groups in the sugar-phosphate backbone — each phosphate carries one negative charge at physiological pH).
• The mass and charge of DNA both scale linearly with length, so charge-per-unit-length is essentially constant. The separation is therefore by size alone.
• When placed in an electric field, DNA fragments migrate towards the positive electrode (anode).
• Smaller fragments move faster and further through the pores of the gel matrix because they encounter less frictional resistance.
• Larger fragments move more slowly because they navigate the pores with more difficulty.
• The result is separation of fragments by size, with the smallest fragments nearest the positive electrode (bottom of the gel as conventionally photographed).

Method

1. Preparing the gel. Agarose powder is dissolved in buffer by heating (typical concentrations 0.7–2% w/v). The molten agarose is poured into a casting tray with a comb to create wells. As it cools (~50°C → room temperature) it sets into a gel with a porous structure. The agarose concentration determines the pore size: higher % gives smaller pores and better resolution of small fragments (1.5–2% for ~100–1000 bp); lower % for larger fragments (0.7% for ~5–25 kb).

2. Loading the samples. DNA samples (PCR products, restriction digests, or fingerprinting reactions) are mixed with a loading dye that contains glycerol (to add density so the sample sinks into the well) and a visible dye such as bromophenol blue (so the migration front can be tracked). Samples are pipetted into the wells.

3. Running the gel. The gel is placed in a tank filled with buffer solution (TAE or TBE) and connected to a power supply. An electric current is applied (typically 80–120 V for 30–90 minutes). The buffer conducts current and maintains a stable pH so that DNA remains negatively charged.

4. Staining and visualisation. After electrophoresis, the gel is stained to make the DNA visible:

   • Ethidium bromide — intercalates between the base pairs of double-stranded DNA and fluoresces orange under UV light. Effective but a known mutagen — handled with gloves; the literature standard for decades.
   • SYBR Safe, SYBR Green, GelRed — safer fluorescent alternatives with similar sensitivity.
   • The gel is photographed under UV (or blue light for SYBR Safe) to create a permanent record.

5. Size determination. A DNA ladder (molecular weight marker) containing fragments of known sizes (e.g. 100 bp, 200 bp, 500 bp, 1 kb, 2 kb, 5 kb, 10 kb) is run alongside the samples. Fragment sizes in the samples are estimated by comparison with the ladder. The relationship between fragment size and migration distance is approximately logarithmic: a plot of log(size) versus migration distance gives a straight line over the linear range of the gel.

Interpreting Gel Electrophoresis Results

• Bands closer to the wells (top of the gel) represent larger fragments.
• Bands closer to the positive electrode (bottom of the gel) represent smaller fragments.
• The intensity (brightness) of a band indicates the quantity of DNA in that band — brighter bands contain more DNA, all else being equal.
• If all copies of a DNA sample are the same size (e.g., a clean PCR product), a single sharp band is seen.
• A restriction enzyme digest of a larger DNA molecule produces multiple bands corresponding to fragments of different sizes — a useful way to verify a recombinant construct or to map a restriction site distribution.

Factors Affecting Migration