SOUTHERN BLOTTING TECHNIQUE

 SOUTHERN BLOT

The southern blot is a way of verifying that a DNA sequense is present in a sample of DNA. This system is named by the British biologist Edwin Southern, its inventor. As detailed below is the protocol for the Southern blot technique:


· RESTRICTION ENDONUCLEASES are used to decrease the high molecular DNA strands into smaller pieces, which are then electrophored on an agarose gel to distinguish them by dimension.


· If the DNA fragments are larger than 15 kb, the acid, such as diluted hCl which purines the DNA fragments can be handled before blotting, breaking the DNA into the smaller pieces so that the gel can be transferred more efficiently from a gel to a membrane.


· The DNA is put in an alkaline solution with (NaOH) to denature the double-stranded DNA when alkaline transference methods are used. Denaturation in an alkaline environment may boost the binding of the loading negatively of the DNA with a positively loaded membrane and separate it into one DNA strand for subsequent hybridization of the probe.


− On top of (or below depending on the direction of transfer) gel, a sheet of nitrocellulose (or nylon) membrane is mounted. Pressure is applied uniformly to the gel to ensure good and even contact between the membrane gel (either with a suction or by putting a pile of paper towels and weights on top of the membrane and gel). The buffer transfer from a high-water area to a low-water area (usually filter paper and paper tissues) via capillary action moves DNA from the gel to the membrane; ion exchange interactions connect DNA with the membrane because of the DNA negative load and the positive membrane charging.


· The membrane is then baked for 2 hours or is exposed to ultraviolet (nylon membrane) radiation in a vacuum or daily oven, and permanently attached to the membrane the transmitted DNA.


· A hybridising probe is then exposed to the membrane — a single DNA fragment with a certain sequence to determine its presence in the target DNA. The DNA probe is marketed so that a flushing or chromog√©nic colouring molecule can normally be detected by adding radioactivity or labelling.


· Excess probe is washed from the membrane after hybridisation and the pattern of hybridization is seen with a radioactive or fluorescent probe in the X-ray film or with the formation of the membrane colour, when a chromogenic detection system is used.




Hybridizing the sensor into a particular DNA fragment on the membrane shows this fragment to contain an additional DNA sequence to the sensor. Transfer of the DNA from electrophoresis gel to a membrane allows simple attachment to the size fractionated DNA of the labelled hybridisation probe. In order to determine the amount of sequences (i.e. gene copies) in the genomes, Southern blots with a restriction of enzyme-digested genomic DNA can be used. A sample which only hybridises to one DNA segment that was not cut by the restrictive enzyme will produce one strip on a Southern blot, while multiple strips can be seen when the sample hybridises to many very similar strands (e.g., those that may be the result of sequence duplication). Modifications in hybridization conditions (i.e., hybridization temperature increases or the concentration of salt decreases) can be used to raise the specificities of a sample and reduce hybridization to less than 100% identical sequences.

Comments

Popular posts from this blog

MORPHOGENESIS

BIOFERTILIZER

VECTORS IN MOLECULAR BIOLOGY