Southern Blot Test: Correct Stepwise Procedure for DNA Analysis

The steps involved in the Southern Blot test should be performed in the following order 1 – x-ray film 2 = electrophoresis 3 = digestion with restriction enzyme 4 = ethidium bromide 5 = radioactive probe
A. 32451
B. 34251
C. 32541
D. 24351

What Is the Correct Order of Steps in the Southern Blot Test?

The correct answer is:

✅ A. 3 → 2 → 4 → 5 → 1 (32451)

The Southern blot test is a molecular biology technique used to detect specific DNA sequences within a mixture of DNA fragments. The process involves multiple steps, each crucial for ensuring accurate detection of the target sequence.

Step-by-Step Process of Southern Blotting

1. DNA Digestion with Restriction Enzymes (Step 3)

   • The first step involves cutting the DNA sample into smaller fragments using restriction enzymes. These enzymes recognize specific sequences and cleave the DNA at those sites, producing restriction fragments of varying sizes.

2. Electrophoresis (Step 2)

   • The digested DNA fragments are then subjected to gel electrophoresis, where they migrate through an agarose gel under an electric field.
   • Smaller fragments travel faster than larger ones, leading to their separation based on size.

3. Staining with Ethidium Bromide (Step 4)

   • The gel is treated with ethidium bromide, a dye that intercalates between DNA bases and fluoresces under UV light.
   • This step allows visualization of DNA fragments to confirm successful electrophoretic separation.

4. Hybridization with a Radioactive Probe (Step 5)

   • The DNA fragments are transferred onto a nitrocellulose or nylon membrane through a capillary or vacuum transfer system.
   • A radioactively labeled DNA probe, complementary to the target sequence, is then introduced. The probe binds to the specific DNA fragment containing the sequence of interest.

5. Detection Using X-ray Film (Step 1)

   • To visualize the hybridized probe, the membrane is exposed to x-ray film (autoradiography).
   • Dark bands appear on the film, indicating the presence of the target DNA sequence.

Why Are the Other Options Incorrect?

• B (34251) and C (32541) – These sequences incorrectly place ethidium bromide or hybridization steps before the correct order.
• D (24351) – Incorrect placement of digestion and staining disrupts the process.

Conclusion

The Southern blot test follows a strict order: DNA digestion, electrophoresis, staining, probe hybridization, and detection. This technique is fundamental in genetic research, forensic science, and medical diagnostics, helping identify gene mutations, hereditary disorders, and viral integrations in DNA.