Lesson 3 of 3 / DNA amplification and analysis
Find a sequence with a probe
How can one band be identified among many DNA fragments?
I can explain Southern blotting and nucleic-acid hybridisation.
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.
A Southern blot is used on a DNA sample. Does it make more copies of the target sequence?
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It finds the target with a probe.
A blot transfers separated DNA to a membrane and detects it by hybridisation. PCR is the method that amplifies.
The key idea
Southern blotting transfers separated DNA to a membrane; a labelled complementary probe detects fragments carrying a matching sequence.
Read the method
Which fragment contains the matching sequence?
Suitable restriction endonucleases first cut the DNA sample into fragments. A gel then separates that mixture by size. A and B both have a 500 bp band, but equal length does not establish equal sequence.
Teal = DNA pattern; brown = labelled probe retained on a complementary target. Stage 4 displays detected probe signal, not the disappearance of all other DNA. Band sizes and sequences are illustrative.
Explanation
The DNA sample is cut into fragments using suitable restriction endonucleases, then separated by gel electrophoresis. The DNA is denatured to single strands and transferred to a membrane while preserving the relative positions of bands. Fixing DNA to the membrane creates a stable pattern for probing.
A labelled single-stranded nucleic-acid probe is incubated with the membrane under suitable conditions. It hybridises by complementary base pairing to matching target DNA. Unbound probe is washed away and the label is detected, revealing the position of target-containing fragments.
The probe sequence provides specificity beyond fragment size alone. Temperature and washing conditions affect which mismatched hybrids remain, so hybridisation conditions matter. A negative result can reflect absent target, failed transfer, unsuitable conditions or insufficient sensitivity rather than a universal absence of all related DNA.
A Southern blot detects DNA, whereas PCR amplifies DNA and a gel separates fragments. These methods can be combined but are not synonyms. A change in a detected fragment's length can indicate a changed restriction site or region size; interpret it using the experimental setup rather than assuming it proves a particular disease.
Step by step
- 1
Preserve the size pattern
Transfer the separated DNA without losing lane relationships.
- 2
Choose a complementary probe
A label reveals where it remains bound.
- 3
Interpret with controls
Separate absence of signal from failure of the method.
Worked example
Work through the evidence
A gel has many bands, but a labelled probe detects only a 2 kb band in one lane. What does this mean?
One way to explain it
A fragment of about 2 kb contains sequence sufficiently complementary to the probe under the conditions used. The other visible bands need not contain that target sequence.
Why this answer works
- Gel staining reveals DNA generally.
- Probe detection identifies a particular complementary sequence.
Watch out for this
A student says: "Southern blotting increases the number of target DNA copies." What is wrong with this?
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It transfers and detects DNA. Amplification is the role of a method such as PCR.
Check your understanding
Why wash the membrane after probe incubation?
Show the answer
To reduce background from unbound or unsuitable probe binding
This makes the detected pattern more specific and interpretable.
Quick recall
How can one band be identified among many DNA fragments?