Question

This is a microbial physiology question related to taxonomy, phylogeny, and physiology:

Can you describe and apply techniques such as: Southern, Western, hybridization, genetic probes, ELISA, TLC, among others?

Answer 1

Southern blotting- is a technique to detect DNA. In this, DNA from the cells is extracted and purified. This is done with restriction enzymes. Various fragments are separated by electrophoresis. Double-stranded DNA is denatured and is transferred to nitrocellulose paper (blotting). Now a labelled probe is added to this paper. Complementary areas get hybridized. The unbound probe is washed off.  The radioautogarph is obtained to check the hybridized areas.

Western blotting- is a technique to detect specific proteins from a complex mixture of proteins extracted from cells. Three approaches are done in this technique-

(1) separation by size,

(2) transfer to a solid support, and

(3) marking target protein using a proper primary and secondary antibody to visualize

Hybridization- hybridization is a phenomenon in which single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal to complementary DNA or RNA. During DNA-DNA hybridization, the DNA of each species is heated to the melting temperature to produce single strands. Then, a strand of DNA from each species is combined and allowed to anneal together or recombine. Finally, the sample is heated if the two combined strands are very similar more hydrogen bonds will unite them meaning a greate temperature or Tm is required to break his union. If the DNA strands are unrelated lower percentage of hydrogen bonding between two strands would be there and a small amount of heat will separate them i.e they will hac=ve lower Tm.

Genetic probe- is a fragment of DNA or RNA of variable length (usually 100-1000 bases long) which is radioactively labelled used in DNA or RNA samples to detect the presence of nucleotide sequences (the DNA target) that are complementary to the sequence in the probe.

ELISA- enzyme-linked immunosorbent assay is a plate-based assay technique designed for detecting and quantifying substances such as peptides, proteins, antibodies and hormones. for example, For an antigen ELISA, antibodies are bound to a plastic surface, a sample is added and if antigens from the virus we are testing for are present they will stick to the antibodies.

TLC-. Thin Layer Chromatography (TLC) is a solid-liquid technique in which the two phases are a solid (stationary phase) and a liquid (moving phase). It is A technique to separate small molecules on a thin film of an adsorbent on glass or semi-rigid plastic surface by their differential mobilities in a liquid phase that passes through the film by capillary action.