Dot Blot Hybridization Technique: Definition, Principle, Procedure and Applications

Definition: Non fractionated or non-electrophoresed samples are directly blotted and immobilized on a nitrocellulose or nylon membrane as dots or spots for hybridization.
This techniques is used to detect the presence of specific sequence of  DNA or RNA in a non-electrophoresed samples
Advantage over Southern blotting:
Sample from different sources can be tested in a single run

Dot Blot Hybridization Technique: Definition, Principle, Procedure and Applications
The key to this method is hybridization.
Hybridization: It is the process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target DNA.
There are 2 important features of hybridization:
  • The reactions are specific-the probes will only bind to targets with a complementary sequence.
  • The probe can find one molecule of target in a mixture of millions of related but non-complementary molecules (same as southern blotting).

Summary of Procedure:
1. Extract and purify DNA or RNA from different sources
2. Apply directly as small dots on nitrocellulose or nylon membrane
3. If DNA, denature it with mild alkali treatment to form single strands
4. Immobilize by baking at 70-80 C for 2-3 H
5. Add labeled probe for hybridization to take place
6. Wash off unbound probe
7. Autoradiograph

Black dots represents samples where target DNA is present or probe has bound
  • Detection of specific DNA or RNA in a sample without the step of electrophoresis
  • Many samples can be screened in a single run

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