Dot Blot Technique

Dot blot technique is a method of identifying the presence of DNA, RNA and Protein in the sample. It is the simplified form of Southern, Northern and Western blotting for the isolation of DNA, RNA and protein respectively.

This method can detect the presence or absence of biomolecule in a single run. Dot blot technique is a very popular method in genetic engineering. This technique can detect the presence of a specific sequence of DNA and mRNA from the transgenic animals or different tissues.

Content: Dot Blot Technique

  1. Definition
  2. Types of Dot Blot Techniques
  3. Advantages
  4. Disadvantages
  5. Applications

Definition

Dot blot technique can define as the process of identification of biomolecules like DNA, RNA or protein to detect its presence or absence in different samples taken from different cells or tissues of the individuals.

Types of Dot Blot Techniques

The dot blot technique is different for the isolation of DNA, RNA and protein. Therefore, based on the isolation method of biomolecules like DNA, RNA and protein, the dot blot techniques are of three types:

DNA Isolation

The identification of DNA by Dot blot technique involves the following steps:

dot blot technique for DNA

Extraction of DNA: In this step, take different samples of the DNA from different tissues or cells.

Blotting: It is the second step which involves the blotting of the different DNA sample directly onto the nitrocellulose or nylon filter membrane.

Denaturation: As the DNA is double stranded so it has to be denatured first to convert it into the single strands by the means of alkali treatment.

Hybridization: After the denaturation process, add the radioactive probe to the filter medium containing a DNA sample. The radioactive probe will bind with the target DNA or hybridize it.

Washing: After hybridization, wash the unbound or free radioactive probe to wash away from the filter medium.

Autoradiography: In autoradiography, expose the filter membrane to the X-ray film through which one can visualize the target DNA.

RNA Isolation

The identification of RNA by Dot blot technique involves the following steps:

dot blot technique for RNA detection

Extraction of RNA: In this step, take different samples of the RNA from different tissues or cells.

Blotting: It is the second step which involves the blotting of the different RNA sample directly onto the nitrocellulose or nylon filter membrane.

Hybridization: Add the radioactive probe to the filter medium containing the RNA sample. The radioactive probe will bind with the target RNA to hybridize it.

Washing: After hybridization, wash the unbound or unhybridized radioactive probe to wash away from the filter medium.

Autoradiography: In autoradiography, expose the filter membrane to the X-ray film through which one can visualize the target RNA.

Protein Isolation

The identification of Protein by Dot blot technique involves the following steps:

dot blot technique for protein detection

Extraction of Protein: In this step, take out the different protein samples from different tissues or cells.

Blotting: Blotting of a protein involves the addition of different protein sample directly onto the nitrocellulose or PVDF filter membrane.

Blocking: Then add bovine serum albumin (BSA) medium or dry milk to block the extracellular space in the filter membrane.

Addition of primary antibody: The primary antibody fixes with the target protein molecule.

Washing: After the binding of the primary antibody with the target protein, wash the filter paper to remove the unbound primary antibody by the PBS buffer.

Addition of secondary antibody: The secondary antibody specifically binds with the primary antibodies. The secondary antibody attaches with the enzymes. Add the substrate after the attachment of the secondary antibody with the primary antibody. The addition of substrate will give a specific colour to the sample.

Colourimetric detection: Measure the intensity of the colour by the colourimeter.

Advantages

  • Dot blot technique does not require the separation of bands on the solid support medium (Agarose) or there is no requirement of electrophoresis.
  • One can detect the presence or absence of genes from the sample of transgenic individuals in a single test run.
  • There is no requirement of transferring of a biomolecule from gel to filter membrane.
  • Dot blot technique involves the direct blotting of biomolecule onto the membrane.

Disadvantages

  • Dot blot method does not give any information about the size and molecular weight of the biomolecules that have to be identified.

Applications

  • Dot blot technique can be used to detect protein concentration.
  • The specific sequence of a gene can be detected in a transgenic individual.

Conclusion

Therefore, the dot blot technique is the method of detecting DNA, RNA and Protein from the different sample will appear at different spots. Each spot will represent one sample. The main principle for the dot blot technique is based on the hybridization method, where a specific radioactive probe will bind with the desired DNA, RNA or protein.

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