Blotting
Molecular Diagnostics
(MDx) Review 564
Principle
Southern blot
Northern blot
Nucleic acids that have been separated by electrophoresis are transferred to solid support membrane.
Used to analyze DNA. Useful for identification of DNA fragments that don’t amplify well, for polymorphisms &
structural alterations.
- DNA extracted from sample.
- DNA cut into fragments with restriction endonucleases.
- Fragments separated by gel electrophoresis.
- dsDNA fragments denatured into single strands by treatment with base.
- Single-stranded fragments transferred (blotted) to solid support medium by capillary action. (Newer methods
use vacuum & pressure to ↑speed of transfer.)
- DNA immobilized by baking or UV light.
- Labeled DNA probe applied.
- Excess probe washed away.
- Visible bands seen where probe hybridized to complementary DNA fragments.
Labor intensive. Rarely used.
Similar method used to analyze RNA. Digestion step isn’t required because RNA is short. Used to determine if a
gene is being expressed by measuring amount of a specific mRNA. Primarily a research tool.
