The phosphodiester backbone at the sites of depurination). The DNA in the gel is exposed to weak acid (which results in partial depurination), followed by strong base (which hydrolyzes The DNA before capillary transfer ( Wahl et al. The problem of dehydration due to lengthy transfer can be alleviated by partial acid/base hydrolysis of This flow reduces the gel to a rubbery substance through which DNA moleculesĬannot easily pass. As elution proceeds, fluid is drawn not only from the reservoir, butĪlso from the interstices of the gel itself. That escape from the gel before it becomes dehydrated. The efficiency of transfer of large DNA fragments is determined by the fraction of molecules Smallįragments of DNA (15 kb in length requires at least 18 h, and even The rate of transfer of the DNA depends on the size of the DNA fragments and the concentration of agarose in the gel. The liquid is drawn through the gel by capillary action, which is established and maintained by a stack of dry, absorbent In upward capillary transfer, DNA fragments are carried from the gel in an upward flow of liquid and deposited on the surface (BACs), and yeast artificial chromosomes (YACs). The techniques described are suitable for SouthernĪnalysis of restriction digests of mammalian genomic DNA but can easily be adapted to accommodate large DNA molecules separatedīy pulsed-field gels, as well as restriction digests of plasmids, cosmids, λ bacteriophages, bacterial artificial chromosomes Many of these improvements have been incorporated into Protocol: Southern Blotting, which deals with transfer of DNA from gels to membranes, and Protocol: Southern Hybridization of Radiolabeled Probes to Nucleic Acids Immobilized on Membranes, which describes hybridization of radiolabeled probes to immobilized DNAs. Use of sensitive phosphorimagers to capture images with high efficiency More efficient blocking agents to prevent nonspecific attachment of radiolabeled probes to membranes ( Church and Gilbert 1984) 1990 Chomczynski 1992), vacuum blotting ( Olszewska and Jones 1988 Trnovsky 1992), bidirectional blotting, and transfer in alkaline buffers ( Reed and Mann 1985)įacile labeling of probes in vitro to higher specific activity ( Feinberg and Vogelstein 1983, 1984) More efficient methods of transfer of DNA from gel to membrane, downward capillary transfer ( Lichtenstein et al. In addition, DNA can now be covalently fixed to the membraneĪfter transfer, eliminating problems caused by leaching of nucleic acids from nitrocellulose membranes during incubation atĮlevated temperatures ( Haas et al. The most significant of these improvements is the use of supported nylon membranes that are far more durable and have a higherīinding capacity than the original nitrocellulose membranes. Increased sensitivity and reproducibility, so that immaculate results are now the general rule rather than the rare exception. However, significant advances over the years in several areas have brought 1976) that they certainly could not be published today. For two or three years after its introduction, the sensitivity of Southern blotting ( Southern 1975) was barely sufficient to detect single-copy sequences in mammalian DNA, and the autoradiographs of the time were so speckledĪnd streaked with background (e.g., see Botchan et al.

Southern hybridization analysis