Southern Blot

By Levi Clancy for Student Reader on
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Southern Blots (aka Southern Transfers) allow researchers to locate DNA fragments of research amidst even millions of restriction enzyme fragments.

Southern Blots usually use conventional DNA probes that bind to sequences even if there are a few mismatched base pairs. Thus, typical Southern Blots only identify mutants with large deletions or insertions. A mutation of only a few base pairs will not be detected, unless that mutation is within a cleavage site. However, there are synthesized oligonucleotide probes -- titled allele-specific oligonucleotides (ASOs) -- that do not bind if even a single base pair is mismatched. By creating ASOs that bind only to wild-type sequences or only to mutant sequences, ASOs are an essential tool in identifying one or two base pair mutations.

Southern Blots are performed in the following steps:
StepOverview

DNA Isolation

DNA can be isolated from any cell except mature red blood cells, which lack nuclei.

DNA Digestion

DNA is broken into fragments by restriction enzymes.

Electrophoresis

Using gel electrophoresis, DNA fragments are separated on the basis of size.

Denaturation

By applying a strong base, double-stranded DNA fragments denature and separate into single strands.

Transfer

A filter paper is placed on top of the gel, and DNA strands transfer from the gel to the filter paper.

Labeling

The filter paper is soaked with single-stranded radioactive probes complementary to genes of interest.

Washing

The filter paper is washed to remove unbound probe.

X-Ray

Shining an x-ray on the filter paper visualizes radioactive probes, revealing where DNA of interest is located.

Interpretation

After finding bands of DNA of interest on the filter paper, those bands can be matched up to the original agarose gel. DNA can then be isolation from specific bands on the agarose gel.