Blotting Techniques Overview

Study Notes

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.

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 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 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.

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).