Question

Give the step-wise procedure of Southern blotting technique. Mention any two important applications of this technique.

Answer 1

The stepwise procedure of Southern blotting technique is as follows:

1. DNA extraction: The first step is to extract DNA from the sample tissue or cells of interest.
2. Restriction enzyme digestion: The extracted DNA is then cut into smaller fragments using specific restriction enzymes.
3. Gel electrophoresis: The fragments are then separated based on their size using gel electrophoresis.
4. Denaturation: The DNA fragments are then denatured using an alkaline solution, which breaks the hydrogen bonds between the two strands, resulting in single-stranded DNA.
5. Transfer: The single-stranded DNA fragments are then transferred from the gel to a nitrocellulose or nylon membrane by capillary action or by applying an electric field.
6. Fixation: The DNA fragments are then fixed to the membrane by either baking it or by UV crosslinking.
7. Hybridization: A labeled DNA probe, complementary to the sequence of interest, is then added to the membrane, which hybridizes with the complementary single-stranded DNA sequence.
8. Detection: The labeled probe bound to the membrane is then detected using autoradiography, fluorescence, or chemiluminescence.

Two important applications of Southern blotting technique are:

1. Identification of specific DNA sequences: Southern blotting can be used to identify specific DNA sequences in a complex mixture of DNA fragments. This technique is widely used in molecular biology and genetics research to identify genes and mutations associated with diseases.
2. DNA fingerprinting: Southern blotting can also be used in DNA fingerprinting to identify individuals based on their unique DNA profile. This technique has important applications in forensic science and paternity testing.