4.1.2Additional lessons
Electrophoresis and PCR — IB Biology HL Revision Notes & Topical Questions
5 min read 5 parts Lesson 28 of 101
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.
Section 1 of 5Gel Electrophoresis
Electrophoresis is used to compare fragments of DNA, RNA or protein.
Principle
- Principle DNA fragments are separated in a gel according to their size when an electric current is passed through it.
Charge on DNA
- Charge on DNA The phosphate groups in the backbone give DNA a negative charge, so fragments move towards the positive electrode, the anode.
The gel
- The gel Agarose gel forms a mesh of pores;
- small fragments pass through easily and move far, whereas large fragments move only a short way.
Preparing samples
- Preparing samples DNA is often cut with restriction enzymes first, loaded into wells at the negative end,
- and run alongside a ladder of fragments of known size.
Visualising
- Visualising After the run, the DNA is stained or labelled with a fluorescent dye so that bands can be seen under suitable light.
Section 2 of 5The Polymerase Chain Reaction
PCR makes millions of copies of a DNA sequence from a tiny sample.
1
The reaction mixture contains template DNA, two primers, free nucleotides, buffer and a heat-stable DNA polymerase, usually Taq polymerase.
2
Denaturation: heating to about 95 °C breaks the hydrogen bonds between the two DNA strands and gives single strands.
3
Annealing: cooling to about 50 to 65 °C lets the short primers bind to complementary sequences at the ends of the target region.
4
Extension: at about 72 °C, Taq polymerase adds nucleotides to the 3′ end of each primer, building a new complementary strand.
5
The cycle is repeated, typically 25 to 35 times, and the number of copies of the target sequence doubles each time.
Section 3 of 5Why Each Component Matters
Why Each Component Matters
Primers
- Primers These short single-stranded DNA sequences mark the start and end of the target and give DNA polymerase a 3′ end to extend.
Taq polymerase
- Taq polymerase Obtained from the bacterium Thermus aquaticus, this enzyme tolerates 95 °C, so it is not denatured during each cycle.
Thermal cycler
- Thermal cycler This machine automatically raises and lowers the temperature through the repeated cycles.
Exponential increase
- Exponential increase Copies double every cycle, so a single molecule gives about a billion copies after thirty cycles.
| Step | Temperature | What happens |
|---|---|---|
| Denaturation | About 95 °C | Strands separate |
| Annealing | About 50-65 °C | Primers bind to template |
| Extension | About 72 °C | Taq polymerase builds new strands |
Section 4 of 5Worked Example
Question
- A PCR starts with 5 copies of the target sequence.
- How many copies are present after 10 cycles, assuming every cycle doubles the number?
- The number of copies = starting copies × 2ⁿ, where n is the number of cycles.
- 2¹⁰ = 1024.
- 5 × 1024 = 5120.
Answer There are about 5120 copies after 10 cycles.
Section 5 of 5Exam Checklist & Tips
4.1.2 – Key Points for the Exam
- Gel electrophoresis Separation of molecules by size in an electric field.
- PCR A technique that copies a DNA sequence many times.
- Primer A short DNA sequence that starts the synthesis of a new strand.
- Taq polymerase A heat-stable DNA polymerase used in PCR.
- Denaturation Breaking of hydrogen bonds between complementary strands.
Common Mistakes
- Saying DNA moves to the negative electrode, when it is attracted to the positive anode.
- Writing that large fragments travel furthest through the gel.
- Confusing the temperatures of annealing and extension.
- Forgetting that PCR needs a heat-stable polymerase.
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