Southern Blotting

SOUTHERN BLOTTING

A Southern blot is a technique for detecting the presence of a DNA sequence in a DNA sample. Edwin Southern, a British scientist, is credited with inventing the approach. The Southern blot technique is described in full below:

• Restriction endonucleases are used to cut high-molecular-weight DNA strands into smaller fragments, which are then separated by size on an agarose gel. • If the DNA fragments are larger than 15 kb, the gel may be treated with an acid, such as dilute HCl, prior to blotting, which depurinates the DNA fragments, breaking the DNA into smaller pieces, allowing more ef


• When using alkaline transfer methods, the DNA gel is immersed in an alkaline solution (including NaOH) to denature the double-stranded DNA. Denaturation in an alkaline environment may increase negatively charged DNA binding to a positively charged membrane, splitting it into single DNA strands for later hybridization to the probe, and eliminates any residual RNA that may still be present in the DNA. A nitrocellulose (or nylon) membrane sheet is placed on top of (or below, depending on the transfer direction) the gel. To guarantee good and even contact between gel and membrane, pressure is given uniformly to the gel (either by suction or by laying a stack of paper towels and a weight on top of the membrane and gel). Buffer transfer by capillary action from a region of high water potential to a region of low water potential (typically filter paper and paper tissues) is used to move the DNA from the gel onto the membrane; ion exchange interactions bind the DNA to the membrane due to the DNA's negative charge and the membrane's positive charge.


• The membrane is then roasted for 2 hours in a vacuum or conventional oven at 80 °C or exposed to ultraviolet radiation (nylon membrane) to permanently bond the transferred DNA to the membrane.


• After that, the membrane is exposed to a hybridization probe, a single DNA fragment with a specified sequence whose presence in the target DNA is confirmed. The probe DNA is labelled to allow detection, typically by adding radioactivity or tagging the molecule with a fluorescent or chromogenic dye.





• Exces probe is rinsed from the membrane after hybridization, and the hybridization pattern is observed on X-ray film using autoradiography in the case of a radioactive or fluorescent probe, or by developing colour on the membrane if a chromogenic detection method is used.


The probe's hybridization to a specific DNA fragment on the filter membrane indicates that this fragment contains complementary DNA sequence to the probe. The transfer of the DNA from the electrophoresis gel to a membrane allows the labelled hybridization probe to easily bind to the size-fractionated DNA. Southern blots using restriction enzyme-digested genomic DNA can be used to count the number of sequences (for example, gene copies) in a genome. A probe that only hybridises to a single uncut DNA fragment will yield a single band on a Southern blot, however several bands are likely to be observed when the probe hybridises to several closely similar sequences (e.g., those that may be the result of sequence duplication). Modification of the hybridization conditions (e.g., increasing the hybridization temperature or decreasing salt concentration) can be used to increase specificity and decrease hybridization of the probe to fewer than 100% identical sequences.

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