Molecular Biology: Southern Blotting Techniques

Blotting & Hybridization Techniques

• Blotting is a technique to transfer DNA, RNA, and proteins onto a carrier for separation, often using gel electrophoresis.

Hybridization Probe

• A probe is a fragment of DNA or RNA (100-10,000 bp) labeled radioactively or fluorescently.
• Hybridization is the binding of single-stranded DNA or RNA to a complementary sequence on another sample DNA or RNA.
• Probes can detect DNA or RNA sequences complementary to the probe's sequence.

Types of Blotting

• Southern Blot: Used to detect DNA.
• Northern Blot: Used to detect RNA.
• Western Blot: Used to detect protein.

Basic Blotting Techniques

• Southern, northern, and western blotting share basic techniques.
• Blotting begins with DNA/RNA/protein separation on a gel.
• Gel bands are transferred to a membrane (nitrocellulose, PVDF, etc.).
• Radiolabeled (or enzymatically labeled) probes bind to the immobilized target.
• Methods detect the molecule of interest.

1. Southern Blotting

• Developed by Sir Edwin Southern in 1975.
• Used to detect specific DNA sequences in samples.
• Key steps:
  • DNA digestion with restriction enzymes.
  • Separation of DNA fragments using electrophoresis.
  • Transfer of DNA to a membrane (e.g., nitrocellulose).
  • Hybridization of a probe to the target DNA fragments.

Procedure of Southern Blotting

• 1. Digest DNA with restriction enzymes.
• 2. Separate DNA fragments by gel electrophoresis.
• 3. Denature DNA fragments.
• 4. Transfer DNA to a nitrocellulose membrane.
• 5. Hybridize the DNA with a labeled probe.
• 6. Wash to remove unbound probe.
• 7. Analyze with an autoradiograph.

Applications of Southern Blots

• Detect specific DNA fragments.
• Isolate specific DNA fragments.
• Identify DNA mutations.
• Identify genetic diseases.
• Identify infectious agents.
• Forensic and paternity testing.

2. Northern Blotting

• Technique to determine the identity, size, and abundance of specific RNA sequences.
• Developed by James Alwine and George Stark.
• Immobilizes RNA molecules on a membrane (nitrocellulose or nylon).
• Uses hybridization techniques to detect specific nucleic acids and genes.

Procedure of Northern Blotting

• 1. Isolate RNA from samples.
• 2. Separate RNA fragments by gel electrophoresis.
• 3. Transfer RNA to a membrane.
• 4. Hybridize the RNA with a labeled probe.
• 5. Wash to remove unbound probe.
• 6. Detect labeled probe (e.g., autoradiography).

Advantages and Disadvantages of Northern Blot

• Advantages: Detects RNA size, relatively inexpensive.
• Disadvantages: Less sensitive than RT-qPCR, sensitive to RNA degradation, requires large amounts of RNA.

Applications of Northern Blot

• Gene expression studies.
• Diagnosis of diseases (e.g., Crohn's disease).
• Detection of viral microRNAs.
• Screening of recombinants.

3. Western Blotting

• Technique used to detect specific proteins in samples.
• Involves separating proteins by gel electrophoresis and transferring them to a membrane.
• Key steps:
  • Denature proteins using SDS.
  • Separate proteins by SDS-PAGE.
  • Transfer proteins to a membrane (nitrocellulose, PVDF, etc.).
  • Incubate membrane with primary antibody specific to the target protein.
  • Detect bound primary antibody with a labeled secondary antibody.
  • Visualize using a substrate reaction (film or digital imager).

Western Blot Procedure

• 1. Sample preparation (lyse, denature).
• 2. Gel electrophoresis (separate proteins by size).
• 3. Transfer proteins to a membrane.
• 4. Antibody detection. (Block membrane, primary antibody, secondary antibody, enzyme detection.)

Use of Western Blotting in Clinical Diagnosis

• Confirms a positive HIV diagnosis.

In Situ Hybridization (ISH)

• Technique to localize specific DNA or RNA sequences within cells or tissues.
• Types include:
  • Fluorescence in situ hybridization (FISH): Fluorescently labeled probes.
  • Chromogenic in situ hybridization (CISH): Chromogen-labeled probes.

Characteristics of ISH Methods

• CISH: Bright field microscopy, visualization of CISH signal and tissue morphology concurrently.
• DNA-FISH: Fluorescence microscopy, multiplex visualization of multiple targets.
• RNA-FISH: Fluorescence microscopy and flow cytometry, simultaneous detection of multiple targets.

Application of ISH

• Prenatal testing (chromosomal abnormalities).
• Pharmacogenomics (drug metabolism).
• Pathogenomics (infectious diseases).