Southern Blotting- introduction, steps, applications, limitations and disadvantages: Southern blotting is a technique which allows the the detection of a specific DNA sequence in a large complex sample of DNA. This technique was named after it inventor and developer, the British biologist, Edward M. Southern in 1975.
Southern blotting is a technique in which transfer of DNA molecule, usually restriction fragments, from an electrophoresis gel to a nitrocellulose or nylon sheet is done in such a way that DNA banding pattern present in the gel is reproduced on the membrane. The restricted DNA might be a plasmid or bacteriophage clone. The key to this method is hybridization, which is a process of forming double stranded DNA molecule between a single stranded DNA probe and a single stranded target patient DNA.
Beffers/ chemicals and equipments required:
- Cell lysis buffer,
- 10X TBE buffer,
- Denaturing buffer,
- Probe buffer,
- Hybridization buffer,
- Washing buffer,
- DNase free water,
- Gel electrophoresis tank,
- Nylon sheet,
- Whatman paper.
Step-1; DNA separation:
In step 1 DNA separation is done. First of all, DNA is extracted from the cells and purified. DNA is extracted with enzyme and a large piece of DNA is chopped into smaller pieces using R.E. Then, this DNA is loaded and run through an agarose gel such that in well 1 DNA marker is added, in lane 2 restricted DNA is added and in well 3 unrestricted (i.e. whole) DNA is added.
Step-2; Blot on membrane:
Two methods are used for transferring DNA to membrane, namely;
- Vacuum blotting,
In vacuum blotting, vacuum is used for the transferring of DNA onto the membrane instead of capillary forces. This process takes about 2 hours and hence is a faster process. The DNA is then further proceeded.
In electro-blotting, electric current is used for the transferring of DNA onto membrane. Here instead of agarose gel polyacrylamide is used due to its high melting point.
Step-3; Labeling with specific probe:
Now, the transferred DNA is attached to the membrane by high temperature or UV light. Then, this blot is incubated with a specific probe.
Step-4; Probe detection:
The unbound prob is washed away from the membrane in 2x SSC buffer and 0.1% SDS. Two types of patterns are used for the detection of hybridization;
- Either it can be done by autoradiography on an X-ray film. This is done in case of radioactive or fluorescent probe.
- Or it can be detected by developing the colour on the membrane, if the probe is chromogenic.
Applications of Southern blotting:
Southern blotting is used in gene discovery and mapping, evolution and development studies, diagnostic and forensic study.
It also determines the molecular weight of restriction fragment and measure their relative amounts in different samples.
Southern blotting is used to analyse the genetic pattern which appear in a person’s DNA.
It also analyses the restriction digestion fragmentation of a DNA or a biological sample.
In genetic analysis of GMOs Southern blotting can be used.
It is also used to detect restriction fragment length polymorphism (RFLP) and variable number of tandem repeats (VNTRs).
Limitations and disadvantages of Southern blotting:
- Southern blotting is more expensive than other methods.
- It is complex and time consuming process.
- It’s another limitation is that it requires large amount of targeted DNA.
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