Southern Blotting: Understanding DNA Fragment Migration in Electrophoresis

Southern blotting: The smaller the DNA fragment….
A. The closer to the origin it will appear
B. The brighter color it produces with ethidium bromide
C. The faster it migrates during separation by electrophoresis
D. The slower it migrates during separation by electrophoresis

Correct Answer: (C) The faster it migrates during separation by electrophoresis

Southern blotting is a widely used molecular biology technique that detects specific DNA sequences within a sample. The process involves DNA digestion with restriction enzymes, gel electrophoresis, membrane transfer, and probe hybridization to identify target DNA fragments.

One of the key steps in Southern blotting is electrophoresis, which separates DNA fragments based on their size.

How DNA Fragment Size Affects Migration in Electrophoresis

During agarose gel electrophoresis, DNA molecules migrate through the gel under an electric field. The movement of DNA is influenced by the following factors:

1. Size of DNA Fragments

   • Smaller DNA fragments travel faster and move farther through the gel.
   • Larger DNA fragments travel slower and remain closer to the origin (wells).

2. Gel Composition

   • Agarose gel concentration affects separation.
   • Higher agarose concentration (e.g., 2%) improves the resolution of small DNA fragments.
   • Lower agarose concentration (e.g., 0.8%) is better for large DNA fragments.

3. Voltage Applied

   • Higher voltage increases migration speed, but excessive voltage can lead to DNA smearing.

Why Option (C) is Correct

Smaller DNA fragments migrate faster through the gel because they experience less resistance from the gel matrix. This makes them move farther from the loading wells compared to larger DNA fragments.

Why Other Options Are Incorrect

• (A) The closer to the origin it will appear ❌

  • Incorrect, because smaller DNA fragments travel farther from the origin, while larger fragments stay closer.

• (B) The brighter color it produces with ethidium bromide ❌

  • Ethidium bromide (EtBr) staining depends on DNA quantity, not size. Larger fragments contain more base pairs, so they bind more EtBr and appear brighter.

• (D) The slower it migrates during separation by electrophoresis ❌

  • Incorrect, because smaller DNA fragments migrate faster, not slower.

Conclusion

In Southern blotting, DNA fragments are separated using electrophoresis, where smaller fragments travel faster through the gel due to reduced resistance. This property is crucial for genetic analysis, disease detection, and forensic studies.