15.5:
Southern Blot
Southern blotting is a technique where a labeled DNA probe hybridizes with a target DNA to detect a particular sequence within a genome.
The technique is named after Edwin Southern, the British biologist who first developed it.
The procedure follows five main steps: electrophoresis, denaturation, membrane transfer, hybridization, and visualization.
First, the genomic DNA is digested by restriction enzymes, and the resulting DNA fragments are loaded on an agarose gel and separated using gel electrophoresis.
For denaturation or separation of the double-stranded DNA into two single-stranded DNA, or ssDNA, the gel is soaked in a sodium hydroxide solution.
The gel is then placed on a sponge in a DNA neutralizing solution containing sodium chloride and tris buffer, to reset its pH to 7.0.
Next, a nylon membrane is placed on top of the gel and weighed down with a stack of paper towels. The ssDNA transfers onto the membrane through capillary action, while the high salt concentration in the neutralizing solution helps the DNA to bind.
When irradiated with UV rays, the DNA becomes covalently cross-linked to the membrane. This prevents diffusion and immobilizes the DNA bands.
The membrane is then soaked in a solution of denatured salmon sperm DNA. The solution coats the membrane and prevents any non-specific binding with the probe DNA.
The probes are short, single-stranded DNA that have a complementary sequence to the target DNA fragments. For visualization, the probes are either labeled with a radioactive phosphorus —P-32, or an enzyme that generates an easily detectable product.
When the membrane is soaked in a buffer solution containing the probes and warmed at 42 °C, the target ssDNA pairs with the complementary probe DNA to form a labeled, hybrid, double-stranded DNA.
After overnight hybridization, the membrane is washed to remove the unbound probes.
For radioactively labeled DNA, the membrane is exposed to an X-ray film for detection.
Enzyme-labeled probes can be detected by adding an appropriate substrate and visualizing the bands as the color or luminescence develops. Because the labeled probes will only bind to the DNA sequence of interest, any visible band indicates presence of the target DNA.
15.5:
Southern Blot
Agarose gel electrophoresis is very useful in separating DNA fragments by size. Running a DNA ladder containing fragments of the known length alongside the sample helps determine the approximate length of the sample DNA fragments. However, additional steps are needed to verify the sequence identity of the sample DNA fragments.
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe – a small ssDNA fragment complementary to the target DNA and labeled with a reporter tag. This process is called Southern blotting.
In Southern blotting, the target DNA is cut into smaller fragments and run on an agarose gel. After denaturing the fragments to yield single strands, the DNA is transferred to a nylon or nitrocellulose membrane. The fragments are then immobilized on the membrane by UV irradiation (for nylon) or heat application (for nitrocellulose). Nylon membranes are more commonly used because of the hazardous nature of baking nitrocellulose membranes in an oven at 80 °C.
Then, the membrane is exposed to a labeled probe. When the probe DNA finds a complementary sequence, they basepair to form a hybrid molecule while the excess unbound probe is washed away from the membrane. This is followed by detection methods, such as autoradiography, to visualize the DNA hybridization patterns.
Southern blots are useful in identifying DNA, determining its size and abundance. Its various applications include investigating changes in genes such as deletions, insertions, or rearrangements. It can also help to determine the number of copies of a gene in a given tissue sample. Because the DNA needs to be fragmented by a restriction endonuclease before being run on a gel, Southern blots can also detect a point mutation in a DNA sequence if it alters a restriction site.
A similar technique called Northern blotting is used to identify RNA sequences in a complex mixture. It is commonly used to detect the expression of a particular gene by assaying for the mRNA transcript.
Leitura Sugerida
- Sambrook, Joseph, Edward F. Fritsch, and Tom Maniatis. Molecular cloning: a laboratory manual. No. Ed. 2. Cold spring harbor laboratory press, 1989.
- Thomas, Patricia S. "[18] Hybridization of denatured RNA transferred or dotted to nitrocellulose paper." In Methods in enzymology, vol. 100, pp. 255-266. Academic Press, 1983.
- Brown, Terry. "Hybridization analysis of DNA blots." Current protocols in molecular biology 21, no. 1 (1993): 2-10.