- 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:
- To detect sequence homology between two DNA molecules without determining the exact base sequence of the molecules.
- Allows identifying particular fragments containing genes of interest.
- To monitor the abundance or size of a particular DNA molecule or to know the length of a restriction fragment.
- Provides a physical map of restriction sites within a gene located normally on a chromosome.
- Reveal the number of copies of genes in the genome.
- Also, reveal the degree of similarity of the gene when compared with the outer complementary genes.
- Can be used to detect the mutation in DNA.
- 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
- Radiolabelling of probes and detecting auto-radiographically by placing the nitrocellulose filter with photographic x-ray film.
- Tagging fluorescent nucleotides to probes.
- 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 180oC 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.
Principle and procedure of Southern blotting