Nucleic acid hybridization Southern blotting

Southern [DNA] (32) blots). Recently, a technique has been described for treating nucleic acid target-probe complexes as antigens in an antigen capture system performed in plastic ELISA plates (38). The latter technique is of interest because it allows automated equipment designed for performing standard ELISA to be used for nucleic acid hybridization techniques. 

Fig. 1. Southern blot analysis of DNA showing (a) step 1, an agarose gel containing separated restriction fragments of DNA, denoted by (—), which is immersed in NaOH to denature the double-stranded stmcture of DNA, and then transferred by capillary flow to a nitrocellulose filter. In step 2, the bound DNA is allowed to hybridize to a labeled nucleic acid probe, and the unbound probe is washed off In step 3, the filter is placed into contact with x-ray film resulting in (b) bands of exposure on the film which are detected after development and correspond to regions where the restriction fragment is...

DNA adsorption properties were first studied using a variety of solid supports for classical analysis methods including Southern and Northern transfers, dot-blotting, colony hybridization and plaque-lifts [31,32]. Studies of the interactions between nucleic acids and nitrocellulose revealed that molecular weight, finite macromolecular conformation, ionic forces and weaker forces of attraction all play a role. DNA is retained on nitrocellulose only in... 

DNA arrays have been categorized into different formats based upon what is immobilized to the surface (also known as the solid phase, substrate, or chip) and what is captured from the sample solution. Definitions change depending upon the format. For the classic Southern dot blot, the sample was first spotted down on the surface, cross-linked, and then bathed with a radio-labeled oligonucleotide under hybridization (complementary nucleic acid strand base-pairing) conditions to detect the presence of a parhcular sequence within the sample. This was called probing. The oligonucleohde... 

The field of DNA microarray has evolved Ifom the key insight of Ed Southern (13), who showed that it is possible to attach nucleic acid to solid support. The resulting Southern blot can be viewed has the first DNA array (14). It was only a small step to improve the technique to filter-based screening of clone libraries, which introduced a one-to-one correspondence between the clone and the hybridization signal (15) in a fixed position in this way the clone could be uniquely identified and information about it accumulated. 

In situ hybridization was derived from the techniques of molecular hybridization of nucleic acids that are isolated from a particular cell population or tissue and bound to solid supports. Hybridization of such averaged membrane-bound nucleic acids identifies different classes of DNA (Southern blot) and RNA (Northern blot). However, ISH fills the gap between the detection of a specific sequence and its precise location within the tissue or the cell (Chevalier et al., 1997). 

Solid phase hybridization is in most cases achieved on membranes. Target nucleic acid is immobilized and subsequently detected by a probe. This approach forms the basis of slot/dot blot hybridization, Northern and Southern hybridization and colony or plaque hybridization. Dot/slot blot hybridization (Kafatos et al., 1979) demonstrates the presence of target sequences but not their size. Although solid phase hybridization is convenient for hybrid/free probe separation, it has the disadvantages that nucleic acid is most often bound noncovalently and that targets are immobilized at fre-... 

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