Lesson 2 of 3 / DNA amplification and analysis
Separate DNA by fragment length
Why does a smaller DNA fragment travel farther in a gel?
I can explain agarose electrophoresis and interpret bands with controls.
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 7 min
Make a guess. You are not marked.
DNA fragments of 500 and 5,000 base pairs are run on a gel. Which travels further from the well?
Show the answer
The 500 base pair fragment.
Both carry negative charge per length. The gel slows longer fragments more, so short ones move further.
The key idea
Negatively charged DNA moves towards the positive electrode; the gel slows longer fragments more, separating sizes.
Read the method
Read direction, size and the negative control
The sample band has mobility matching approximately 500 bp. This supports a product of that size, not proof of its exact sequence. The blank NT lane supports a clean run, while the positive lane supports a working assay.
L = ladder; + = positive control; S = sample; NT = no-template control. Sizes are in base pairs. Original illustrative gel, not real experimental data; band distance is not linearly proportional to length.
Explanation
DNA carries negative charge from its phosphate backbone. Samples are loaded into wells near the negative electrode and an electric field drives DNA towards the positive electrode. Agarose acts as a molecular sieve, so shorter fragments generally migrate farther than longer ones under the same conditions.
A DNA ladder contains fragments of known sizes. Compare sample bands with the ladder to estimate length; a band close to a known marker suggests a similar size. Migration distance is not simply proportional to base-pair length, so use an appropriate calibration if a precise estimate is required.
A band represents many DNA molecules with similar mobility, not one molecule visible to the naked eye. Staining or fluorescent detection makes DNA detectable. Band brightness can suggest relative DNA amount under controlled conditions, but saturated images and fragment length complicate direct quantitative comparisons.
A band at the expected size supports the presence of a product of that approximate length. It does not by itself establish its exact base sequence: unrelated fragments can share a length. Negative and positive controls, specific probes or sequencing can strengthen identification. In laboratory use, follow supervision for stains, electric equipment and any illumination hazards.
Step by step
- 1
Read the lane labels
Identify ladder, controls and samples first.
- 2
Read direction
Locate wells and positive electrode.
- 3
Make a limited inference
Estimate size without claiming exact sequence.
Worked example
Work through the evidence
A sample band lies between 500 bp and 1000 bp ladder bands, nearer 500 bp. What can be concluded?
One way to explain it
It is consistent with a fragment between those sizes, closer in mobility to the 500 bp marker. Its exact sequence cannot be identified from this position alone.
Why this answer works
- Use the ladder for scale.
- Do not interpolate linearly in base pairs without a suitable calibration.
Watch out for this
A student says: "A brighter band must always contain longer DNA." What is wrong with this?
Show the answer
Position mainly reflects size; brightness mainly reflects detected amount and imaging conditions.
Check your understanding
Where should DNA generally move from the wells?
Show the answer
Towards the positive electrode
The phosphate backbone gives DNA a negative charge.
Exam-style questions
Check your understanding
Which statement about agarose gel electrophoresis of DNA is NOT correct?
Show the answer
Longer fragments travel further from the wells
The gel slows longer fragments more, so shorter fragments travel further.
Quick recall
Why does a smaller DNA fragment travel farther in a gel?