BY: SREELAKSHMI (MSIWM012)
Blotting is that the technique used for the transferring of nucleic acids or proteins by immobilizing them onto a solid support generally nylon or nitrocellulose membranes. Blotting of macromolecule is that the central technique for hybridization studies. Macromolecule labeling and hybridization on membranes have formed the thought for a selection of experimental techniques involving understanding of phenomenon, organization, etc. Identifying and measuring specific proteins in complex biological mixtures, like blood, have long been important goals in scientific and diagnostic practice. More recently the identification of abnormal genes in genomic DNA has become increasingly important in clinical research and guidance. Blotting techniques are done to identify unique proteins and macromolecule sequences. They have been developed to be highly specific and sensitive and became important tools in both biology and clinical research. Generally principle behind the blotting methods are fairly simple and typically contains four separate steps: electrophoretic separation of protein or of macromolecule fragments within the sample; transfer to and immobilization on paper support; binding of analytical probe to concentrate on molecule on paper; and visualization of bound probe. Molecules during a sample are first separated by electrophoresis then transferred on to an easily handled support medium or membrane. This immobilizes the protein or DNA fragments and they provides a faithful replica of the first separation, they also facilitates subsequent biochemical analysis. After being transferred to the support medium the immobilized protein or macromolecule fragment is localized by the utilization of probes, like antibodies or DNA, that specifically bind to the molecule of interest. Finally, the position of the probe which is suppose bound to the immobilized target molecule is visualized usually by autoradiography.
SOUTHERN BLOTTING TECHNIQUE
Southern Blot is the analytical technique which is utilized in biology, immunogenetics and other molecular methods to detect or identify DNA of interest from a mixture of DNA sample or a specific base sequence within a strand of DNA.The technique was in 1975 by a biologist E.M.Southern for analyzing the related genes during a DNA fragment and thus named as Southern blotting in his honor.
Principle of southern blotting
The process involves the transfer of electrophoresis-separated DNA fragments to a carrier membrane which is usually nitrocellulose and thus the next detection of the target DNA fragment by probe hybridization. Hybridization refers to the tactic of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target DNA. Since the probe and target DNA are complementary to each other, the reaction is restricted which aids within the detection of the precise DNA fragment.
The gene of interest is isolated from the DNA employing a restriction endonuclease .by the action of restriction endonuclease the DNA gets into small fragments during which the specified fragment is additionally present. In order to seek out the proper the fragment agarose gel electrophoresis is performed. Supported the relative molecular mass of strands it get separated during electrophoresis. As we’ve used the double stranded DNA The separated strands also are double stranded it’s converted to single strand the gel is treated with mild alkali like NaOH .It helps in breaking the chemical bond between the double stranded DNA the obtained single stranded DNA is transferred to membrane with the assistance of a transfer solution .IN a tray transfer solution is kept, a glass is kept for the support of gel. On top a nitrocellulose membrane is employed alongside some papers .Above all a weight is kept. Due to the capillarity transfer solution transfers the strands to membrane. To fix the DNA on membrane heat treatment or UV rays are provided. Now the membrane is incubated in a probe solution. Probes are short oligonucleotide sequence used for the detection of molecules. Probes contains radiolabeled molecules which are complementary to the gene of interest. The probes get attached to the DNA strands. This strands are often visualized as using autoradiography to understand whether the gene of interest got transferred to the membrane or not. Southern blotting is additionally called as macromolecule hybridization. As the probe which may be a single strand and therefore the single stranded DNA gets hybridized.
• Identifying specific DNA during a DNA sample.
• Making of RFLP (Restriction Fragment Length Polymorphism) maps
• Detection of mutations, deletions or gene rearrangements in DNA
• For criminal identification and DNA fingerprinting (VNTR)
• Detection and identification of Trans genes for transgenic individual
• Mapping of restriction sites
• For diagnosis of infectious diseases
• Prognosis of cancer and diagnostic procedure of genetic diseases
• Determination of the relative molecular mass of a fragment and to live relative amounts in several samples.