Plant Tissue Culture is a technique of growing cells, tissues or organs in sterilized nutrient media under controlled aseptic conditions. The plant material to be cultured may be cells, tissues or plant organs such as excised root tip, shoot tip, shoot bud, leaf petiole, inflorescence, anther, embryo, ovule or ovary. Continue reading What is Plant Tissue Culture?
Explants are small pieces of plant parts or tissues that are aseptically cut and used to initiate a culture in a nutrient medium. Explants can be taken from different parts of a plant such as shoots, leaves, stems, flowers, roots, and from many types of mature cells provided they are able to de-differentiate into totipotent cells. Continue reading What are Explants?
Southern Blotting is an elegant technique that can be used to identify the locations of genes and other DNA sequences on restriction fragments separated by gel electrophoresis. This technique is named after the Edwin Southern (1975) who developed it.
The important feature of this technique is the transfer of the DNA molecules that have been separated by gel electrophoresis onto nitrocellulose or nylon membranes. This can be carried out by following these steps:
The genomic DNA isolated from cells/tissues is digested with one or more restriction enzymes.
This mixture of DNA fragments is loaded into a well in an agarose or polyacrylamide gel and then subjected to electrophoresis.
The gel containing separated DNA molecules is soaked in an alkali (e.g. NaOH) to denature the dsDNA (double stranded) in order to make it single stranded. Then, the gel is transferred to a place where a nitrocellulose membrane can be placed onto this. In this step, the ssDNA (single stranded) fragments get bound to the nitrocellulose membrane or nylon filter by capillary action.
These DNA fragments are fixed onto the membrane by heating it to 80°C. This practice provides a more permanent record of the sample since DNA begins to diffuse out of the gel if it is left as such for few hours.
The conditions of hybridization, including the temperature and salt concentration, are critical for this process to take place effectively. This is because conditions are specific for each DNA probe and for each sample of DNA.
Now, the membrane can be treated with a labeled DNA molecule, for example, a Gene Probe. A gene probe may be
- Purified RNA.
- A segment of cloned DNA.
This single-stranded probe will hybridize under the right conditions to complementary fragments immobilized onto the membrane. A series of washing steps with buffer is carried out to remove any unbound probe.
At last, the membrane is exposed to X-Ray film to develop an autoradiograph. After the film is developed, the dark bands show the precise location of DNA sequences that have hybridized with the probe.
Applications of Southern Blotting:
- An invaluable method for gene analysis and confirming DNA cloning results.
- Used in DNA Fingerprinting or Forensic labs to detect minute quantities of DNA (to identify Parenthood, thieves, rapists etc.)
- Used to detect the presence of specific genes among different species or strains of a single species (referred to as Zoo Blotting).
- Highly useful for the determination of Restriction Fragment Length Polymorphism (RFLP) associated with pathological conditions.