In recent times, the people of India have been heard talking about gene sequencing, gene editing, gene sequencing, and many other similar terms. It seems like, they are now getting introduced to the idea and principle behind personal genomics. Continue reading Personal Genomics: Gene Sequencing & Analysis in India.
A most common and important source of genetic variability is known to be present uniformly throughout the genome is termed Single Nucleotide Polymorphisms or SNPs. Interest in SNPs lies in the fact that these polymorphisms may be responsible for the differences in disease susceptibility, drug metabolism and response to environmental factors between individuals. Even, if they are not directly responsible for the disease, they serve as genetic markers for a nearby locus that might be responsible. Continue reading What are SNPs? Why are Scientists interested in them?
An average Human cell (diploid) contains about 6.4 billion base pairs of DNA divided among 46 chromosomes. The length of each base pair is about 0.34 nm. Therefore, if the DNA molecule in a diploid cell were laid out end to end, the total length of DNA would be approximately 2 meters. Continue reading How a 2 meters long DNA is fitted into a 2 micrometers Nucleus?
It is a well-known fact that DNA acts as a genetic material in most of the organisms on this planet earth. However, it is also clear that RNA also acts as a genetic material, but only in some viruses (for example, Tobacco Mosaic Viruses, QB Bacteriophage, etc.).
Continue reading Why Nature Preferred DNA over RNA?
The discovery that genetic information is coded along the length of DNA was one of the major achievements of science in the 20th century. This polymeric molecule, DNA, is the chemical basis of heredity and is the fundamental units of genetic information.
Continue reading Who discovered that DNA is the Genetic Material?
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.