U01.01.042 Blotting Procedures

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Learning Objectives

  • Differentiate between Southern, Northern, and Western blots.
  • Identify the specific target molecules and probes used in each technique.
  • Apply the SNoW DRoP mnemonic for rapid identification.
  • Recognize clinical applications, such as diagnosing Fragile X syndrome.

1. The SNoW DRoP Mnemonic

To remember which blot corresponds to which molecule, use this classic cross-match table:

Blot Type Target Molecule
Southern DNA
Northern RNA
Western Protein

2. Southern Blot (DNA)

Used to identify the size and presence of specific DNA sequences.

  1. DNA is enzymatically cleaved into smaller fragments.
  2. Fragments are separated by gel electrophoresis and transferred to a membrane.
  3. The membrane is exposed to a labeled DNA probe that anneals to its complementary strand.
  4. Visualization occurs via film or digital imaging.

Clinical Use: Determination of heterozygosity or counting CGG repeats in the FMR1 gene to diagnose Fragile X syndrome.

3. Northern Blot (RNA)

Similar to Southern blotting, but the target is an RNA sample.

  • Purpose: Studying mRNA levels (gene expression) and size.
  • Utility: Highly effective at detecting splicing errors.

4. Western Blot (Protein)

This technique shifts from nucleic acids to proteins.

  • Process: Proteins are separated by electrophoresis and transferred to a membrane.
  • Probe: A labeled antibody is used to bind the specific protein of interest.
  • Result: Helps identify the specific protein and determines its quantity.

Activity