Chapter summary
DNA amplification and analysis, at a glance
Scan the key ideas, or hide the answers and try to recall them.
01
PCR amplifies a chosen DNA region
What makes PCR selective rather than copying every molecule equally?
Key idea and reminders
Two primers define a target, and repeated denaturation, annealing and extension amplify it under suitable conditions.
- Denature, anneal, extend.
- Primers flank the target with 3-prime ends facing inward.
- Ideal 2^n growth is an assumption, not a perpetual law.
Keep in mind: Ordinary targeted PCR needs appropriate primers matching known or sufficiently characterised flanking regions.
02
Separate DNA by fragment length
Why does a smaller DNA fragment travel farther in a gel?
Key idea and reminders
Negatively charged DNA moves towards the positive electrode; the gel slows longer fragments more, separating sizes.
- DNA moves towards the positive electrode.
- Shorter fragments usually travel farther.
- Same size does not prove same sequence.
Keep in mind: Position mainly reflects size; brightness mainly reflects detected amount and imaging conditions.
03
Find a sequence with a probe
How can one band be identified among many DNA fragments?
Key idea and reminders
Southern blotting transfers separated DNA to a membrane; a labelled complementary probe detects fragments carrying a matching sequence.
- Restriction-digest, separate, denature, transfer, probe, detect.
- Hybridisation depends on complementary pairing.
- Southern detects target DNA at a band position.
Keep in mind: It transfers and detects DNA. Amplification is the role of a method such as PCR.