Blotting Techniques Overview

Blotting Techniques

• Blotting techniques are used for analyzing DNA, RNA, and proteins
• The lecture discusses Southern, Northern, Western, and Dot blot techniques.

Southern Blotting

• Southern blotting is the first blotting technique, making analysis and recording easy.
• It identifies DNA fragments with specific sequences.
• DNA fragments are separated by electrophoresis, then transferred to nitrocellulose, and hybridized with a 32P-labeled single-stranded DNA probe.
• The fragment containing the sequence is then visualized by autoradiography
• Procedure:
  • DNA sample is digested with a restriction enzyme and electrophoresed.
  • DNA bands in the gel are denatured into single strands using an alkali solution.
  • The gel is placed on top of a buffer-saturated filter paper on a solid support (e.g., glass plate).
  • A nitrocellulose filter membrane is placed on top of the gel and a stack of papers.
  • Capillary action pulls the buffer through the gel, the transfer medium, and up through the paper towel stack.
  • Nitrocellulose membrane with single-stranded DNA bands is baked at 80°C for 2-3 hours to permanently fix DNA.
  • DNA fragments are probed for the sequence of interest using hybridization.
  • The membrane is then washed to remove unbound DNA.
  • X-ray film is exposed to the membrane to get autoradiographs.

Southern Blotting Applications

• Extremely sensitive for mapping restriction sites around a single copy gene sequence in a complex genome (e.g., human).
• Useful for forensic purposes when using mini-satellite probes for identifying minute DNA amounts.
• Used to analyze the role of DNA methylation in gene expression.
• Used for detecting mutated genes in genetic disorders.

Northern Blotting

• Northern blot procedure is similar to Southern blotting, but RNA is analyzed instead of DNA.
• RNA species are separated by size using electrophoresis through agarose gel with formaldehyde or glyoxal and dimethyl sulfoxide.
• Separated RNA bands are blotted onto chemically reactive filter paper.
• RNA species are then hybridized to radiolabeled DNA probes.
• Auto radiography is used to locate RNA that's complementary to the probe.
• Applications:
  • Used for detection and quantitative estimation of hybridized mRNA.
  • Used to study RNA degradation, RNA half-life, and RNA splicing.
  • Useful in studying gene expression.

Western Blotting

• Western blotting is used for detecting proteins with particular specificity.
• When a transferred gene expresses in transformed cells, the translated protein product can be identified by this technique.
• Procedure:
  • Proteins are isolated or extracted.
  • Extracted proteins are subjected to polyacrylamide gel electrophoresis (PAGE), and transferred onto nitrocellulose.
  • Radiolabeled specific antibody is added to the membrane, binding only to complementary proteins.
  • Antibody is labeled with 125I, and the signal is detected by autoradiography.
    • If radiolabeling isn't used, a second antibody tagged with an enzyme can be used.

Western Blotting Applications

• HIV confirmatory test detects anti-HIV antibody in human serum.
• Definitive test for Bovine spongiform encephalopathy (BSE, "mad cow disease").
• Confirmatory test for Hepatitis B infection.

Dot Blot Technique

• Used to detect the presence of a given sequence of DNA/RNA (non-fractionated).
• DNA from many samples can be tested in a single test.
• Simplification of Northern, Southern, and Western blotting methods; biomolecules aren't separated by electrophoresis.
• Procedure:
  • A mixture containing the molecule to be detected is applied directly as a dot on a membrane.
  • The dot is spotted through circular templates on the membrane or paper.
  • Detection is done by nucleotide probes (for Northern/Southern) or antibodies (for Western).

Dot Blot Applications

• Detecting Chlamydia-trachomatis infection and other STDs.
• Detecting anti-diacyltrehalose antibodies in Tuberculosis and Typhoid Fever patients.