Analysis of DNA by Southern Blotting

Upward Capillary Transfer

In upward capillary transfer, DNA fragments are carried from the gel in an upward flow of liquid and deposited on the surface of the solid support (Southern 1975). The liquid is drawn through the gel by capillary action, which is established and maintained by a stack of dry, absorbent paper towels (see Fig. 1). The rate of transfer of the DNA depends on the size of the DNA fragments and the concentration of agarose in the gel. Small fragments of DNA (<1 kb) are transferred almost quantitatively from a 0.7% agarose gel within 1 h; larger fragments are transferred more slowly and less efficiently. For example, capillary transfer of DNAs >15 kb in length requires at least 18 h, and even then the transfer is not complete. The efficiency of transfer of large DNA fragments is determined by the fraction of molecules that escape from the gel before it becomes dehydrated. As elution proceeds, fluid is drawn not only from the reservoir, but also from the interstices of the gel itself. This flow reduces the gel to a rubbery substance through which DNA molecules cannot easily pass. The problem of dehydration due to lengthy transfer can be alleviated by partial acid/base hydrolysis of the DNA before capillary transfer (Wahl et al. 1979; Meinkoth and Wahl 1984). The DNA in the gel is exposed to weak acid (which results in partial depurination), followed by strong base (which hydrolyzes the phosphodiester backbone at the sites of depurination). The resulting fragments of DNA (∼1 kb in length) can then be transferred rapidly from the gel with high efficiency. The depurination reaction must not proceed too far; otherwise, the DNA will be cleaved into small fragments that are too short to bind efficiently to the solid support. Depurination/hydrolysis can also cause the bands of the final autoradiograph to assume a “fuzzy” appearance, presumably because of increased diffusion of DNA during transfer. Therefore, depurination/hydrolysis is recommended only when it is known ahead of time that the target DNA fragments will exceed 15 kb in length.

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Figure 1.

Upward capillary transfer of DNA from agarose gels. Buffer drawn from a reservoir passes through the gel onto a stack of paper towels. DNA, eluted from the gel by the moving stream of buffer, is deposited onto a nitrocellulose or nylon membrane. A weight applied to the top of the paper towels helps to ensure a tight connection between the layers of material used in the transfer system.

Downward Capillary Transfer

In downward capillary transfer, DNA fragments are carried in a downward direction in a flow of alkaline buffer and are deposited onto the surface of a charged nylon membrane. Various arrangements of wicks, reservoirs, and different formulations of transfer buffers have been described to achieve downward transfer (e.g., see Lichtenstein et al. 1990; Chomczynski 1992). In our hands, the best results are achieved using 0.4 m NaOH and a setup in which the transfer buffer is drawn from reservoirs to the top of the gel through wicks and pulled through the gel by an underlying stack of paper towels. Transfer of DNA fragments is rapid, and the intensity of signal is ∼30% greater than can be achieved by conventional upward transfer. This improvement probably results from a more efficient migration of DNA fragments through the interstices of the gel, which is not under pressure from weights placed on top.

Simultaneous Transfer to Two Membranes

In simultaneous transfer to two membranes, when the target DNA fragments are present in high concentration (e.g., in restriction digests of cloned DNAs), the capillary method can be used to transfer DNA simultaneously and rapidly from a single gel to two nitrocellulose or nylon membranes. The only source of transfer buffer is the liquid trapped in the gel itself, and thus the efficiency of transfer is relatively poor. This method is not recommended when high sensitivity is required (e.g., detection of single-copy sequences in mammalian DNA), but it is perfectly adequate for Southern analysis of plasmids, bacteriophages, or cosmids or the genomes of simple organisms (e.g., Saccharomyces cerevisiae and Drosophila) by Southern hybridization. Too little mammalian genomic DNA is transferred by this method to allow signal detection routinely.

Electrophoretic Transfer

The electrophoretic transfer method is not practical when nitrocellulose is used as the solid support because the high-ionic-strength buffers required to bind nucleic acids to nitrocellulose conduct electric current very efficiently. In consequence, it is necessary to use large volumes to ensure that the buffering power of the system does not become depleted by electrolysis. In addition, extensive external cooling is required to overcome the effects of ohmic heating.

Electrophoretic transfer has undergone a recent resurgence with the advent of charged nylon membranes and has become the method of choice for analysis of small fragments of DNA separated by electrophoresis through polyacrylamide gels (Stellwag and Dahlberg 1980; Church and Gilbert 1984). Nucleic acids as small as 50 bp will bind to charged nylon membranes in buffers of very low ionic strength (Reed and Mann 1985).

Although single-stranded DNA and RNA can be transferred directly, fragments of double-stranded DNA must first be denatured in situ, as described in Protocol: Southern Blotting [Green and Sambrook 2021a]. The gel is then neutralized and soaked in electrophoresis buffer (e.g., 1× TBE) before being mounted between porous pads aligned between parallel electrodes in a large tank of buffer. The time required for complete transfer depends on the size of the fragments of DNA, the porosity of the gel, and the strength of the applied field. However, because even high-molecular-weight nucleic acids migrate relatively rapidly from the gel, depurination/hydrolysis is unnecessary, and transfer is generally complete within 2–3 h. Because electrophoretic transfer requires comparatively large electric currents, it is often difficult to maintain the electrophoresis buffer at a temperature compatible with efficient transfer of DNA. Many commercially available electrophoretic transfer machines are equipped with cooling devices, but others are effective only when used in a cold room.

Vacuum Transfer

In vacuum transfer, DNA and RNA can be transferred rapidly and quantitatively from gels under vacuum. Several vacuum transfer devices are now commercially available in which the gel is placed in contact with a nitrocellulose or nylon membrane supported on a porous screen over a vacuum chamber. Buffer, drawn from an upper reservoir, elutes nucleic acids from the gel and deposits them on the membrane.

Vacuum transfer is more efficient than capillary transfer and is extremely rapid. DNAs that have been partially depurinated and denatured with alkali are quantitatively transferred within 30 min from gels of normal thickness (4–5 mm) and normal agarose concentration (<1%). If performed carefully, vacuum transfer can result in a twofold to threefold enhancement of the hybridization signal obtained from Southern transfers (Olszewska and Jones 1988).

All of the commercially available apparatuses work well as long as care is taken to ensure that the vacuum is applied evenly over the entire surface of the gel. Special care should be taken with the wells of horizontal agarose gels, which tend to break during preparation of the gel for transfer. If this occurs, the wells should be trimmed from the gel before transfer. (The wells need not be trimmed from the gel as long as they are unbroken.) It is also important not to apply too much vacuum during transfer. When the vacuum exceeds 60 cm of water, the gels become compressed, and the efficiency of transfer is reduced.