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.
Copyright © 2024 MyJoVE Corporation. All rights reserved