Principle and procedure of Southern blotting

Introduction

• It is a method developed by E.M Southern (1975).
• It combines the use of restriction endonuclease, electrophoresis, and DNA probes for analyzing the related genes in a DNA restriction fragment.
• It is used for various purposes. They are:

1. To detect sequence homology between two DNA molecules without determining the exact base sequence of the molecules.
2.  Allows identifying particular fragments containing genes of interest.
3. To monitor the abundance or size of a particular DNA molecule or to know the length of a restriction fragment.
4. Provides a physical map of restriction sites within a gene located normally on a chromosome.
5. Reveal the number of copies of genes in the genome.
6. Also, reveal the degree of similarity of the gene when compared with the outer complementary genes.
7. Can be used to detect the mutation in DNA.
8. Colony blotting i.e. a version of southern blots can be used to identify clones from other species that are related to any gene of interest.

Image source: microbenotes

Principle of southern blotting

• It actually localizes the DNA separated by electrophoresis.
• The cut DNA are separated by gel electrophoresis in this technique.
• The length of the fragment (expressed as base pairs) is calculated by comparing the position of the band relative to the standard fragment of known size.
• The gene of interest is only one of the number of fragments.
• The fragments are soaked in alkali to denature the DNA and transferred to positively charged membranes such as nylon or nitrocellulose filter.
• Fragments adhere to the membrane creating a blot or imprint, the position of the fragment being comparable to that of electrophoresis.
• The bound DNA is incubated with a probe DNA containing a sequence complementary to a sequence within a gene of interest.
• It is done at salt concentration and temperature close to that at which nucleic acid denatures and renatures.
• The probe DNA will only hybridize to its exact complement.
• A higher molar excess of probes also favors hybridization.
• The hybridized probe can be detected by

1. Radiolabelling of probes and detecting auto-radiographically by placing the nitrocellulose filter with photographic x-ray film.
2. Tagging fluorescent nucleotides to probes.
3. Labeling probes by enzymes such as alkaline phosphatase and horse radish peroxidase, so that change in color or emission of light occurs with the addition of substrate

• The pattern observed on the southern blot depends upon the specific restriction enzyme and probe used.

Procedures of southern blotting

• DNA is firsts digested with restriction enzyme to produce fragments of unequal length.
• The digested sample is then subjected to gel electrophoresis which results in the separation of DNA molecules based on their size.
• DNA bands in the gel are denatured into single strands by alkali treatment.
• The gel is led on the top of a buffer saturated filter paper, the edge of filter paper being immersed in buffer.
• The above preparation is placed on the solid support (glass plate).
• A sheet of nitrocellulose membrane placed on the top of the gel and a stack of paper towels (dry filter papers) is laid on top of the membrane.
• Weight of 0.5 kg is kept on top of all.
• The buffer is drawn up by filter paper wick, passes through the gel to a nitrocellulose membrane, and finally to paper towels.
• Buffer carries with it the single-stranded DNA. SS DNA (negative charge) binds nitrocellulose membrane (positive charge).
• It is then left overnight.
• The nitrocellulose membrane with SS DNA blotted in it.
• It is then baked at 180^oC for 2-3 hours to fix DNA permanently.
• The membrane has replica bands from the agarose gel.
• Then the membrane is placed in a solution containing radiolabelled RNA or denatured DNA probe of known sequences.
• These are complementary in sequence to the blot transferred DNA.
• The radiolabeled nucleic acid probe hybridizes the complementary DNA on nitrocellulose filter.
• Membrane is then washed thoroughly to remove unbound DNA or probe.
• X-ray film is exposed to hybridized membrane to get auto radiographs.
• In this way, southern blotting is performed without missing a single step.

References:

i) https://microbenotes.com/southern-blot-principle-steps-and-applications/

ii) https://www.genome.gov/genetics-glossary/Southern-Blot

Principle and procedure of Southern blotting