Bacterial Conjugation – A Primitive form of Sexual Reproduction.

Several Bacteria (like Escherichia Coli) exhibit a form of sexual reproduction called Bacterial Conjugation. It is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells. It was discovered by Nobel Prize winners, Joshua Lederberg and Edward L. Tatum in 1946.

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Euglena – A Protist with Flexible Lifestyle

Euglena is a large Genus of unicellular Protists, which belongs to phylum, Euglenaceae. Thousands of species of Euglena are present in ponds, lakes, streams and also in the waterlogged area such as marshes. Continue reading Euglena – A Protist with Flexible Lifestyle

What is Southern Blotting?

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:

1. Cleavage:

The genomic DNA isolated from cells/tissues is digested with one or more restriction enzymes.

2. Electrophoresis:

This mixture of DNA fragments is loaded into a well in an agarose or polyacrylamide gel and then subjected to electrophoresis.

3. Blotting:

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.

4. Hybridization:

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.
  • cDNA.
  • 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.

5. Autoradiography:

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.

What is Cell Theory?

The concept that the cell is the basic unit of life is known as the Cell Theory. It is one of the most fundamental generalizations of biology and ranks with Charles Darwin’s theory of organic evolution and T.H. Morgan’s theory of the gene of modern biology. Continue reading What is Cell Theory?