SOUTHERN BLOTTING

Southern blotting is a well known technique used in modern biology for the detection of a single specific DNA sequence (RFLPs) in a in the highly complex mixture of DNA fragments ranging from few to that of whole human genome .this technique was devised by Edwin Southern, A British biologist .Similar methods for RNA and Proteins are named northern and western blotting techniques respectively.

NEED

For obtaining a specific DNA sequence the Genome is first cut into smaller size fragments produced by digestion with the help of restriction endonuclease that cuts the DNA at specific sites. For whole genome there could be large amount of fragments so these are separated though Agarose gel electrophoresis .By gel electrophoresis the DNA fragments are separated on the basis of their size ,the smallest fragment travel the farthest through the gel towards the positive electrode and largest remain near the well. In a complex mixture, many fragments will have the same or nearly the same length and thus migrate together during electrophoresis. So instead of a band we observe a smear. Here lies the need of probing for identification and separation of a specific fragment within one of the bands  by hybridization but since the probe(labeled complementary DNA sequence) do not readily diffuse into the original gel so we require to first denature it with alkali and transfer onto a nitrocellulose filter or nylon membrane by blotting. And this procedure is termed as southern blotting.

Synopsis Need Detailed procedure Applications Drawback References

DETAILED PROCEDURE

With the help of restriction enzymes like BamH1, Hind III, EcoRI which cuts the DNA at specific sites we get many fragments of various lengths .These are separated on the basis of molecular weight with the help of gel electrophoresis. Where it is forced to run through Agarose gel in the presence of electric field, DNA being negatively charged moves towards the positive electrode and the smaller moves the faster. These fragments are made fluorescent by staining the gel with ethidium bromide.  We generally obtain bands of DNA, but in case of large amount of fragments we often get smears of DNA.

This gel is soaked in alkali like NaOH for denaturation or conversion into single stranded DNA for probe binding.

The denaturation in an alkaline environment provides for improved binding of the negatively charged DNA to a positively charged membrane and destroys any residual RNA that may still be present in the DNA.

This gel setup is placed in a trough containing buffer and on a plate covered with filter paper whose ends are immersed in the buffer solution. a sheet of nitrocellulose membrane or nylon is placed over the gel. To apply pressure a stack of paper towels are placed over it then a glass sheet and finally some weight for proper and even contact between gel and membrane. The DNA binds to the membrane by ion exchange interaction since the DNA is negatively charged and membrane has positive charges

For making the binding permanent the membrane is exposed to high temperature (60-100 °C) or ultraviolet radiation so the DNA cross links covalently to the membrane. Thus we obtain an exact replica of the fragments as on original gel, like the clones in a library.The membrane is then incubated under hybridization conditions with a specific 32P-radio-labeled single-stranded complementary DNA strand, which usually is generated from a cloned restriction fragment. The DNA restriction fragment that is complementary to the probe hybridizes, and location of the restriction-fragment-probe duplex on the membrane can be revealed by autoradiography

A particular fragment in the midst of a million others can be readily identified in this way, like finding a needle in a haystack.

APPLICATIONS

In human genome and other eukaryotes there are many repetitive DNA (short sequences repeated thousands of times in tandem) which can be easily detected by the probe. This can be helpful in identification of a organism since there are particular number of repeats that varies among individuals (except between identical twins)

With a suitable probe, the pattern of bands produced by DNA fingerprinting is distinctive for each individual. Combining the use of several probes makes the test so selective that it can positively identify a single individual in the entire human population. So this technique finds its use as a potent weapon in forensic sciences.

DRAWBACK

The Southern blot procedure requires relatively fresh DNA samples and larger amounts of DNA. So its sensitivity is enhanced by using PCR to amplify vanishingly small amounts of DNA. This allows investigators to obtain DNA fingerprints from a single hair follicle, a drop of blood, a small semen sample from a rape victim, or samples that might be months or even many years old.

REFERENCES

Lehninger: Principles of biochemistry

Lodish: Molecular and cell biology

Stryer: Biochemistry