Lesson 1 of 3 / DNA amplification and analysis
PCR amplifies a chosen DNA region
What makes PCR selective rather than copying every molecule equally?
I can explain PCR ingredients, thermal cycles, advantages and limitations.
I can explain PCR, gel electrophoresis, Southern blotting and nucleic acid hybridisation.Syllabus
Syllabus 9477, Core 2(j). Explain PCR, gel electrophoresis, Southern blotting and nucleic acid hybridisation.
About 8 min
Make a guess. You are not marked.
You start PCR with 10 copies of a target sequence and run 20 ideal cycles. Roughly how many copies do you get?
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About ten million.
Each ideal cycle doubles the target: 10 x 2^20 is about 1 x 10^7.
The key idea
Two primers define a target, and repeated denaturation, annealing and extension amplify it under suitable conditions.
Read the method
Which way can each primer extend?
Heat separates the DNA strands by disrupting base pairing. The covalent backbones remain intact. Typical teaching value: about 95 C.
Blue = template DNA; brown = primers; green = newly extended DNA. Arrowheads show synthesis direction, not movement of a whole strand.
Starting with 5 target copies: 5 x 2^3 = 40 under perfect doubling. This is an arithmetic model; real amplification efficiency and early product lengths can differ.
Explanation
PCR requires template DNA, two suitable primers, free deoxyribonucleotides, thermostable DNA polymerase and an appropriate buffer with required ions. The primers bind opposite strands with their extendable 3-prime ends facing towards the target region.
Heating denatures the double-stranded DNA by separating base pairs. Cooling to a suitable annealing temperature allows primers to pair with complementary sequences. At an extension temperature suited to the polymerase, new strands grow 5-prime to 3-prime from the primers. Repeating these stages allows newly made DNA to become template.
Under ideal doubling, a starting amount N becomes approximately N x 2^n after n cycles. Real reactions eventually deviate as reagents become limiting, products accumulate and amplification efficiency changes. The earliest products can extend beyond the target; defined-length products become dominant over cycles.
PCR is rapid and sensitive and needs little starting DNA, but contamination can generate false positives. Primer design requires suitable flanking-sequence information, inhibitors can block amplification, and ordinary PCR alone does not reveal the complete sequence or prove that a detected organism is alive. Include a no-template negative control and a known suitable positive control.
Step by step
- 1
Define the target
Choose primers on opposite strands.
- 2
Explain each temperature stage
Separate strands, bind primers, extend DNA.
- 3
Check the controls
Distinguish contamination from failure to amplify.
Worked example
Work through the evidence
Starting with 5 target copies, what is the ideal amount after 8 doubling cycles?
One way to explain it
5 x 2^8 = 1280 copies, under the ideal doubling assumption.
Why this answer works
- Apply the exponent to the cycle count.
- Actual efficiency and the definition of target-length molecules can affect observed yield.
Watch out for this
A student says: "PCR can amplify any unknown sequence without primers or sequence information." What is wrong with this?
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Ordinary targeted PCR needs appropriate primers matching known or sufficiently characterised flanking regions.
Check your understanding
A no-template control produces the expected band. What is the main concern?
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Contamination or a non-specific product must be investigated.
A positive negative-control lane undermines the interpretation of sample bands.
Exam-style questions
Check your understanding
A PCR starts with 5 copies of a target. After 10 ideal cycles, about how many copies are there?
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About 5,000
5 x 2^10 = 5 x 1,024, which is about 5,000. Each ideal cycle doubles every copy.
Quick recall
What makes PCR selective rather than copying every molecule equally?