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4.1.2Additional lessons

Electrophoresis and PCR — IB Biology SL Revision Notes & Topical Questions

5 min read 5 parts Lesson 100 of 142

📚 IB Biology SL Hub / Topical Revision
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Curriculum Equation Key قوانين الرقم الهيدروجيني والاتزان الأيوني - pH & Acid-Base Equilibrium FormulasFast Exam Solution
pH = -log[H⁺] | pOH = -log[OH⁻] | pH + pOH = 14 | [H⁺][OH⁻] = 10⁻¹⁴
Word Equation & Conditions: الرقم الهيدروجيني = -لوغاريتم تركيز أيون الهيدروجين | pH + pOH = 14
Location & Specifications: الاتزان الأيوني، كيمياء 3 ثانوي، التحصيلي، و AP Chemistry.
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Part 1 of 5Gel Electrophoresis

Electrophoresis separates charged molecules such as DNA, RNA and proteins according to their size and charge.

1
An agarose gel containing small wells is set in a tank and covered with a buffer solution that conducts electricity.
2
DNA samples mixed with a loading dye are placed into the wells with a micropipette, with the wells at the negative electrode end.
3
A direct current is applied, and the negatively charged DNA, because of its phosphate groups, moves through the gel towards the positive anode.
4
Short fragments move easily through the pores of the gel and travel further than long fragments in the same time.
5
The separated fragments are made visible with a fluorescent dye under ultraviolet light or with labelled probes, producing a pattern of bands.
FactorEffect on movement
Net chargeNegative DNA moves towards the anode
Fragment lengthShorter fragments move faster and further
Gel pore sizeSmaller pores slow large molecules more

Part 2 of 5The Polymerase Chain Reaction

Purpose
  • Purpose PCR copies a specific target sequence many times in a test tube, producing enough DNA for analysis from a tiny sample.
Ingredients
  • Ingredients The mixture contains template DNA, two primers, free nucleotides, a heat-stable DNA polymerase and a buffer.
Taq polymerase
  • Taq polymerase Taken from the bacterium Thermus aquaticus, it is not denatured by the high temperature used to separate DNA strands.
Primers
  • Primers Short single-stranded DNA sequences that bind to the ends of the target and give polymerase a starting point.
Thermal cycler
  • Thermal cycler A machine that automatically repeats the heating and cooling cycles, each lasting a few minutes.

Part 3 of 5The Three Stages of Each Cycle

The Three Stages of Each Cycle
Denaturation
  • Denaturation The mixture is heated to about 95°C, which breaks the hydrogen bonds between the two strands to give single strands.
Annealing
  • Annealing The temperature is lowered to roughly 50–60°C so that primers bind to complementary sequences on the template strands.
Extension
  • Extension At about 72°C Taq polymerase adds nucleotides to each primer, building a new complementary strand in the 5′ to 3′ direction.
Exponential growth
  • Exponential growth Every cycle doubles the number of target molecules, so n cycles from one molecule give 2ⁿ copies.

Part 4 of 5Worked Example

Question

  • A single DNA molecule is amplified by PCR through 30 cycles.
  • Assuming every cycle doubles the number of molecules perfectly, calculate the number of copies and compare it with 10 cycles.

  1. Number of copies after n cycles = 2ⁿ.
  2. After 10 cycles: 2¹⁰ = 1 024 copies.
  3. After 30 cycles: 2³⁰ = 1 073 741 824 copies.
  4. Ratio = 2³⁰ ÷ 2¹⁰ = 2²⁰ = 1 048 576.

Answer About 1.07 × 10⁹ copies after 30 cycles, around one million times more than after 10 cycles.

Part 5 of 5Exam Checklist & Tips

4.1.2 – Key Points for the Exam

  • Gel electrophoresis A technique that separates molecules in an electric field according to size and charge.
  • Anode The positive electrode, towards which negatively charged DNA moves.
  • PCR Polymerase chain reaction, an in vitro method that amplifies a chosen DNA sequence.
  • Primer A short single-stranded DNA sequence that anneals to the template and starts copying.
  • Taq polymerase A heat-stable DNA polymerase from Thermus aquaticus used in PCR.

Common Mistakes

  • Saying DNA moves towards the negative electrode; DNA is negatively charged and moves to the anode.
  • Thinking longer fragments travel further; shorter fragments move faster through the gel.
  • Forgetting to name the enzyme and say why it must be heat stable.
  • Stating PCR needs a living cell; it is carried out in vitro in a tube.

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