Vacuum blotting enhances nucleic acid transfer

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Atm'11988, VoL 4, no. 4

Vacuum blotting enhances nucleic acid transfer

The capillary blotting technique originated by Southern~ constituted a revolution in the analysis of genome organization and expression. In this method the gel is soaked in alkali for between 30 minutes and one hour to Eva Olszewska and Ken Jones denature the DNA fragments, and is then equilibrated in neutrafizing soluNucleic acids can be transferredfrom an agarose gel to a transfer membrane using tion for a similar period before being capillary Southern blotting or e!ectroblotting techniques. Vacuum blotting represents placed on the transfer apparatus. an alternative method that offers a number of advantages. This consists of a supporting plate covered with a sheet of filter paper saturated in a transfer buffer and kept wet by means of paper wicks dipping into a reservoir after alkaline treatment before being sandwiched of the same buffer. The filter is suitably masked with together with the transfer membrm'le in a cassette, which plastic film to form a window which directs the upward is then mounted between parallel platinum electrodes in a capillary flow of buffer through the gel, which is placed buffer tank. Transfer takes about two hours and is over it. Mobilized DNA is then trapped on the transfer quantitatively more efficient than capillary blotting. In membrane placed on the upper gel surface. Capillary flow addition, since high molecular weight bands are mobilized through the gel is maintained by placing a weighted stack faster, depurination is unnecessary. However, electroblotting is less convenient to set up, involves more of absorbent paper towels on top of the membrane. Sm~ll DNA fragments of I kb or less transfer from an complex and expensive apparatus, with provision for 0.8% agarose gel within two hours, whereas fragments external cooling to compensate for the ohmic heating greater than 15 kb take 15 hours or mor," This tends to result in underrepresentation of higher molecular weight fragments. This relatively long time required for preeO treatment and transfer, which permits some diffusion of DNA, significantly reduces band resolution. More rapid and even transfer may be obtained by cleaving DNA E ~ molecules in the gel by a 15 minute acid depurination step • lw, after electrophoresis and before denaturation. However, >'~ this obviously also increases the rate of DNA diffusion during subsequent steps. Diffusion of fragments in the reverse direction to the capillary flow significantly 10 20 30 40 50 60 70 reduce~ the DNA available for hybridization, resulting in Vacuum= cm H20 further loss of information. However, this has sometimes been exploited to obtain two blots simultax~eously by placing transfer membranes on both sides of the gel. ! ~.

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Electroblotting Southern first suggested improvements to the conventional method~, using a high voltage to obtain a more rapid and unidirectional transfer of nucleic acids. In electroblotting the pretreatment steps are slightly altered in that the gel is equilibrated in transfer buffer for about two hours Reservoir

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Fig, 1, Exploded diagrammatic side view of blotting appar.~tus. This consists of an upper rectangular open frame which constitutes the reservoir. This is clamped via an O-ring to a box-shaped vacuum chamber which has a sheet of porous polyethylene filter inset in its top. A sheet of polythene (mask) with a suitably sized window to accommodate the transfer membrane is clamped between the upper and lower sections. The vacuum chamber is fitted with a vacuum inlel. See text for further details.

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Fig, 2, The effect of vacuum on DNA transfer efficiency. Paired end-labelled restriction digests of X phage DNA were transferred to nylon membranes (upper set) from 1% agarose gels (lower set) at the vacuum stren9ths indicated for 30 minutes. Membranes and gels were then autoradiographed to monitor transfer, which is optimal between 3050 cm H20. Above 60 cm H20, gel collapse impedes DNA transfer.

effect of the electric current, and uses lm'ge volumes of buffer. Stray voltage fields in some commercially available apparatus can generate artefactual DNA bands which are revealed when the blot is hybridized with an abundant-sequence probe. Vacuum blotting The idea of using a vacuum to speed up the transfer h~ gel blotting occurred both to us and to others, as evidenced by the recent appearance of vacuum blotting devices on the market. The basic design of such devices (~) 1988, E l s e v i e r Publications, C a m b r i d g e

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T I G - Apd11988, Vol. 4, no. 4 23.1

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Fig. 3. A comparison of transfer of identical amounts of endlabelled ~. phage EcoRI and Hindlll restriction fragments under optimal conditions in each of three blotting methods. Results monitored by autoradiographing filters. The times taken for transfer and pretreatment are indicated in each case. The transfer speed, quantitative recovery and resolution of transferred fragments are clearly superior in the case of vacuum blotting.

The efficiency of transfer is affected by a number of parameters. (1) Vacuum level. Sufficient vacuum must be applied to prevent movement of the gel during pretreatment and transfer, but vacuum levels (measured by water gauge) above 70 cm H20 progressively compress the gel and immobilize the DNA, thus reducing transfer. Optimal transfer is obtained over a fairly broad range of 30-60 cm H20 (see Fig. 2). (2) Transfer time. As little as five minutes may be required to t ~ s f e r more than 50% of the DNA from a gel. Transfer of a wide molecu!~_r-weight range of fragments is essentially complete after 30 minutes and longer times lead to no further removal of the trace of DNA remaining in the gel. (3) Gel thickness and concentration. The results described so far were obtained with 4 mm thick 1% agarose gels, which are in the normal practical range. However, 1% agarose gels of up to I cm in thickness can be successfully transferred at the optimal vacuum conditions established above. When thicker gels are used, the transfer time may need to be slightly increased. Gel strengths raLging from 0.6% to 1.2% give satisfactory transfers.

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(Fig. 1) consists of a base module which incorporates a combined vacuum chamber and solution trap plus a porous polyethylene support screen, and an upper assembly which constitutes the buffer reservoir. A polythene film mask, with a window of the same dimensions as the transter membrane but slightly smaller than the gel, is clamped between the two sections. An O-ring forms a fluid- and vacuum-tight seal. When the membrane is placed in the window and the gel placed over the window, fluid in the upper reservoir can only enter the fluid trap by passing through the gel and transfer membrane. Open sample wells are placed so that they overlap the edge of the window in the polythene mask by a millimetre or so in order to ensure a proper vacuum. In the case of horizontal gels, the wells can be placed over the tr~sfer membrane if they do not penetrate the entire thickness of the gel. The origin position can then be marked either on the mask or on the transfer membrane as appropriate.

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Gel pre-treatment in situ Depurination, denaturation and equilibration of agar= ose gels can be performed in situ on the vacuum blotting apparatus within 10 minutes. Another advantage is that very little fluid norm~y passes through the gel during denaturation or transfer by this technique; for example, the fluid contained in a wetted sheet of Whatman filter paper placed on the gel may be enough to keep the gel surface wet and ensure transfer.

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The vacuum produced by a water-driven vacuum pump human DNAs with a hypervariable sequence probe after is usually too high for optimal transfer and, due to electrophoresis and transfer by three blotting methods using fluctuating water pressure, too variable. Either an elec- their respective optimal performance conditions. Upper row trically driven vacuum pump modified to produce an of autoradiographs shows signal after ovemight exposure. appropriate range of stable vacuum levels, or one of the Lower row shows signal after three days exposure. Vacuum commercially avail"able devices such as the LKB Vacu- blotting rapidly reveals features which are not detected after much lelger exposures using the other two methods. Gene TM,yields better results.

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(4) Transfer membranes. All currently available membrane types (including pure nitrocellulose or nylon based) give good results when used under the conditions recommended by the manufacturers. In the case of nylon membranes, for example Hybond (Amersham), excellent transfer can be obtained using either 20 x SSC or distilled water. Comparative resolution and efficiency of different blotting methods From a comparison of capillary, electro- and vacuum blotting under optimal conditions for each, it is clearly apparent that vacuum blotting results in a marked improvement in band resolution (Fig. 3). Transfer was also tested by hybridization after vacuum blotting 7.5 pg of Alul-dlgested electrophoresed human DNA. The probe used was one which detects repetitive sequences over a wide size range of restriction fragments. Recovery of DNA after vacuum blotting was significantly greater than after capillary or electroblotring, as determined by the difference in hybridization signal after overnight exposure (Fig. 4). Even three days' exposure failed to reveal comparable detail in conventional blots. Vacuum blotting should therefore permit cost savings to be achieved by, for example, either using less DNA for transfer or using probes of lower specific activity. Northern vacuum blotting Transfer of RNA from agarose gels to hybridization

April 1988, VoL 4, no. 4

membranes, usually referred t~ as northern blotting, is also rapidly and conveniently carried using the vacuum transfer apparatus, with the same advantages found for DNA.

Summary To summ.arize, vacuum blotting has several significant advantages over existing techniques. The transfer of nucleic acids is rapid and quantitative with excellent band resolution over a wide range of molecular weights. DNA recovery is significantly improved, thus considerably facilitating detection of rare sequences ~ a t may be lost by the use of previous methods. The preparative stages before transfer can be carried out rapidly without further handling of the gel and consequent risk of damage. Very little fluid is used at any stage, thus effecting considerable economies in costs and time.

Acknowledgements We thank Mrs Deirdre Hay for technical assistance. We are grateful to LKB Producter AB for providing a VacuGeneTM vacuum transfer apparatus for evaluation in these experiments.

References I Southern,E. M. (1975)]. Mot. Biol. 98, 503-517 2 Arnheim,N. and Southern, E. M. (1977)Cell 11, 363-370 Eva Olszewska and Ken Jones are in the Department of Genetics, University of Edinburgh, Edinburgh EH8 9YL, UK.

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