HOME
The Info List - Southern Blot


--- Advertisement ---



(i) (i) (i)

A SOUTHERN BLOT is a method used in molecular biology for detection of a specific DNA sequence
DNA sequence
in DNA samples. Southern blotting combines transfer of electrophoresis -separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization .

The method is named after the British biologist Edwin Southern
Edwin Southern
who first published it in 1975. Other blotting methods (i.e., western blot , northern blot , eastern blot , southwestern blot ) that employ similar principles, but using RNA or protein, have later been named in reference to Edwin Southern's name. As the label is eponymous , Southern is capitalised, as is conventional for proper nouns . The names for other blotting methods may follow this convention, by analogy.

CONTENTS

* 1 Method * 2 Result * 3 Applications * 4 See also * 5 References * 6 External links

METHOD

* Restriction endonucleases are used to cut high-molecular-weight DNA strands into smaller fragments. * The DNA fragments are then electrophoresed on an agarose gel to separate them by size. * If some of the DNA fragments are larger than 15 kb , then prior to blotting, the gel may be treated with an acid, such as dilute HCl . This depurinates the DNA fragments, breaking the DNA into smaller pieces, thereby allowing more efficient transfer from the gel to membrane. * If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution (typically containing sodium hydroxide ) to denature the double-stranded DNA. The denaturation in an alkaline environment may improve binding of the negatively charged thymine residues of DNA to a positively charged amino groups of membrane, separating it into single DNA strands for later hybridization to the probe (see below), and destroys any residual RNA that may still be present in the DNA. The choice of alkaline over neutral transfer methods, however, is often empirical and may result in equivalent results. * A sheet of nitrocellulose (or, alternatively, nylon ) membrane is placed on top of (or below, depending on the direction of the transfer) the gel. Pressure is applied evenly to the gel (either using suction, or by placing a stack of paper towels and a weight on top of the membrane and gel), to ensure good and even contact between gel and membrane. If transferring by suction, 20X SSC buffer is used to ensure a seal and prevent drying of the gel. Buffer transfer by capillary action from a region of high water potential to a region of low water potential (usually filter paper and paper tissues) is then used to move the DNA from the gel onto the membrane; ion exchange interactions bind the DNA to the membrane due to the negative charge of the DNA and positive charge of the membrane. * The membrane is then baked in a vacuum or regular oven at 80 °C for 2 hours (standard conditions; nitrocellulose or nylon membrane) or exposed to ultraviolet radiation (nylon membrane) to permanently attach the transferred DNA to the membrane. * The membrane is then exposed to a hybridization probe —a sing