This is a detailed description of how 2D gel replicon analysis was carried out in our laboratory several years ago and is still carried out in some cases. We recommend that the reader also consult the more recent modification of these techniques by Soo-Mi Kim. For further information, we recommend consulting the following publications:
1. Nawotka, K.A. and Huberman, J.A. (1988) Two-Dimensional Gel Electrophoretic Method for Mapping DNA Replicons. Mol. Cell. Biol. 8: 1408-1413.
2. Huberman, J.A., Spotila, L.D., Nawotka, K.A., El-Assouli, S.M. and Davis, L.R. (1987) The In Vivo Replication Origin of the Yeast 2µm Plasmid. Cell 51: 473-481.
3. Brewer, B.J. and Fangman, W.L. (1987) The Localization of Replication Origins on ARS Plasmids in S. cerevisiae. Cell 51: 463-471.
4. Brewer, B.J., Sena, E.P. and Fangman, W.L. (1988) Analysis of Replication Intermediates by Two-dimensional Agarose Gel Electrophoresis. Cancer Cells, Vol. 6, Eukaryotic DNA Replication (Kelly, T. and Stillman, B., eds.), pp. 229-234, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
5. Huberman, J.A. and Nawotka, K.A. (1988) Mapping Eukaryotic Replication Origins. Cancer Cells, Vol. 6, Eukaryotic DNA Replication (Kelly, T. and Stillman, B., eds.), pp. 225-228, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
6. Linskens, M.H.K. and Huberman, J.A. (19990) Ambiguities in results obtained with 2D gel replicon mapping techniques. Nucl. Acids Res. 18: 647-652.
7. Huberman, J.A. (1994) Analysis of DNA replication origins and directions by two-dimensional gel electrophoresis. In: The Cell Cycle: A Practical Approach, Fantes, P. and Brooks, R. F. (eds.), Oxford University Press, pp. 213-234.
8. Friedman, K.L., and Brewer, B.J. (1995) Analysis of replication intermediates by two-dimensional agarose gel electrophoresis. Meth. Enzymol. 262: 613-627.
9. Huberman, J.A. (1997) Mapping replication origins, pause sites and termini by neutral/alkaline two-dimensional gel electrophoresis. Methods: A Companion to Methods in Enzymology 13: 247-257.
The same first dimension gel may be used for both neutral/neutral and neutral/alkaline 2D analysis. Because the two techniques provide independent, complementary information, we strongly recommend using both techniques in unknown, exploratory situations. In our experience, firm conclusions can be reached much more rapidly when information is provided by both techniques. However, for repeated analyses of replication efficiency at known origins, the neutral/neutral technique is preferred, because it is somewhat simpler and considerably more sensitive. The following instructions are written assuming that both neutral and alkaline second dimension gels will be run.
The first dimension gel should separate replicating molecules according to mass (that is, according to extent of replication). The conditions of first dimension electrophoresis should be chosen to minimize dependence of mobility on structure. For this reason, low gel percentages and low voltage gradients are recommended. Our standard technique involves the use of a 20 X 20 cm 0.4% agarose gel, prepared in TAE buffer (supplemented with 0.1 µg/ml ethidium bromide), with 22 wells across the top, each well having a cross section of 5 mm X 1-2 mm. Due to focussing at the well/gel boundary, the resolution obtained with the 2 mm well width is as good as with the 1 mm width. The gel may be formed as thick as necessary to accommodate the sample volume, up to a thickness of 1 cm. Usually our gels are about 5 mm thick, with 1-2 mm agarose under the wells and a well depth of 3-4 mm. Such a gel will accommodate a volume of 10 to 30 µl of sample, depending on the depth of agarose under the wells and on the thickness of the wells. We include a low concentration of ethidium bromide so that the DNA bands can be visualized immediately after conclusion of electrophoresis without having to wait to stain the gel. In our experience, this does not lead to detectable damage to replicating molecules during the run; note, however, that our runs are carried out overnight with room lights off. It is a good idea, if using ethidium bromide in the first dimension gel, to minimize exposure of the gel to light.
The samples should be prepared so that the final DNA concentration does not exceed 200 µg/ml. For analysis of single-copy yeast sequences, we get good signals in neutral/alkaline gels with 500 ng2 µg of caffeine-wash DNA (see our protocol for BND-cellulose fractionation). Note: the BND-cellulose fractionation scheme leads to some loss of replicating molecules. In some situations (for example, when analyzing a slow replication process [slow replication fork movement leads to accumulation of replicating molecules]) it may not be necessary to use BND-cellulose fractionation. About 5 µg of unenriched log phase S. cerevisiae or S. pombe genomic DNA per well usually provides satisfactory signals from single copy sequences in neutral/neutral gels. The samples should be prepared so that their final ionic composition is similar or identical to that of the electrophoresis buffer. This is good electrophoretic practice in general; it minimizes distortion of the electric field in the vicinity of the sample wells, which smears bands. We use 2.5-4% Ficoll to increase sample density; in our experience Ficoll yields sharper bands than sucrose or glycerol.
We run multiple samples in separate lanes of the first dimension gel. At least one lane should contain marker DNAs to cover the size range of interest. Sometimes this requires separate lanes for low molecular weight and high molecular weight markers. One lane should contain the DNA intended for the neutral second-dimension gel, and another lane should contain the DNA intended for the parallel alkaline second-dimension gel. To prevent possible cross contamination between samples, there should be an empty lane on each side of each lane containing DNA intended for second-dimension analysis.
The first dimension should be run long enough so that the molecular weight region of interest is well resolved, but not so long that the region of interest won't fit into the available space in the second dimension gel. In practice, we carry out submerged gel electrophoresis at 16 to 20 V (for a 20 cm long gel) overnight, using TAE buffer supplemented with 0.1 µg/ml ethidium bromide, with the intention that molecules of 1 kb should migrate about 8 cm from the origin.
If large restriction fragments (8-15 kbp) are being analyzed, modification of first and second dimension gel conditions may be required. See the papers from the Calos and Méchali labs which are referenced in the 1993 Huberman chapter in The Cell Cycle: A Practical Approach. For the first dimension, the following conditions have been found in our lab to give improved display of large restriction fragments: 0.35% agarose in TBE, room temperature, no ethidium bromide, run about 24 hrs at about 0.75 V/cm. Although not tested, I suspect that use of TAE would provide even better resolution of large fragments and that the use of low concentrations of ethidium bromide would have no significant effect on resolution.
One can determine when first dimension gel electrophoresis is complete by examining the gel under 360 nm UV light (360 nm should be used to minimize nicking). As soon as the marker DNAs have migrated the desired distance, the electrophoresis is stopped, and the first dimension gel is photographed using 360 nm light. The lanes of interest are then cut out with a clean razor blade, scalpel, or sharpened spatula Do not use an ordinary spatula because it may produce a rough edge (leading to blurring of signal because the 0.4%-1% agarose interface, where "stacking" occurs, will not form a sharp line). For the same reason, it's likely to be helpful to use a ruler as a cutting guide for the cuts along the long sides of the lanes. Only 360 nm light should be used while cutting out lanes, for the reason stated above. The lanes should be cut so that there is little or no non-DNA-containing agarose on each side of the lane, and each lane should be no longer than 1/2 the length of the preparative comb (in our case, each lane should be no longer than 8.5 cm), so that each second dimension gel can hold 2 first dimension lanes (see the accompanying modified protocol by Soo-Mi Kim for analysis of up to 4 first-dimension lanes across a second-dimension gel, with up to 12 lanes total per gel!).
The second dimension gels can be prepared while the first dimension gel is running. For the neutral second dimension, we use a modification of the conditions of Brewer and Fangman (Cell 51: 463-471, 1987). These conditions are designed to emphasize the dependence of mobility on DNA structure through use of high percentage agarose, low run temperature, high voltage, and high concentration of ethidium bromide. We use a 20 X 20 cm 1% agarose gel prepared in 1 X TBE supplemented with 0.5 µg/ml ethidium bromide. For the alkaline second dimension, we use a 20 X 20 cm 1% agarose gel prepared in water, since agarose will not gel under alkaline conditions. After insertion of the first dimension lane (see below), the gel is equilibrated with alkaline electrophoresis buffer (see below). Note that the use of 1% agarose for both second dimension gels permits remarkable sharpening of the DNA bands in the second dimension due to "stacking" of the DNA molecules at the interface between the 0.4% first dimension lane and the 1% second dimension gel.
Both the neutral and alkaline second dimension gels are formed using preparative well combs which, in our case, produce a long preparative well (17 cm X 1-2 mm) flanked by two small marker wells (2.5 X 1-2 mm).
After the second dimension gels have solidified, the wells should be enlarged so that the first dimension lanes can be inserted into them. Using a sharp instrument, cut away the agarose above the preparative well in the second dimension gel. The cuts can extend all the way to the top of the gel, leading to the formation of a large rectangular hole:
Next, carefully transfer the appropriate first dimension lanes into the hole. It's good to have a convention for placement of these lanes, such as always placing the high-molecular-weight end of the lane on the left. The bottom of each first dimension lane should be placed directly on the exposed surface of the gel trough so that it will also be on the bottom in the second dimension gel. Don't try standing the first dimension lanes on their edges; it won't work, and, in any case, the stacking effect at the 0.4%/1% gel boundary will produce sharp second dimension bands, even from a gel lane that was initially over 5 mm wide! The first dimension lanes should be placed parallel to the bottom edge of the preparative well, but not up against that edge or bubbles may be trapped between the two gels. Leave about 2 mm space between the two gels.
After the first dimension lane(s) have been properly placed, use a microwave oven to melt sufficient 0.4% agarose in TAE buffer to fill in the space around the first dimension lane(s). This can be fresh agarose or can come from a portion of the first dimension gel that did not contain sample. The purpose of using "first dimension" agarose to fill in the space around the first dimension lane is to ensure that the 0.4%/1% agarose interface occurs at the perfectly horizontal line formed by the preparative well comb (note the difference in Soo-Mi Kim's protocol, in which a preparative well comb is not usually used). Make sure the agarose is thoroughly melted and thoroughly mixed. Let it cool somewhat, to about 65° C, then pour or pipette it quickly (but gently) into the spaces surrounding the first dimension lanes. Add enough agarose to thoroughly cover the first dimension lanes, so that they will be firmly held in place in the final solid gel. Important: use a spatula or other instrument to remove any bubbles that may be trapped between the first and second dimension gels. Optional: at this point, with liquid agarose and no air bubbles between the first and second dimension gels, the first dimension gel may be moved (by pressure from a spatula or other instrument) so its bottom edge is abutted against the top edge of the second dimension gel. That top edge will be very smooth because it was formed by the preparative comb. Important: work quickly so that the fill-in gel can be completely poured, all bubbles can be removed, and the first dimension lanes can be properly positioned before the gel begins to solidify. It would probably be a good idea to practice these manipulations with some dummy gels before attempting them with gels containing precious experimental material.
After the agarose has solidified, place the neutral second dimension gel into its electrophoresis box and fill the buffer troughs with 1 X TBE buffer, supplemented with 0.5 µg/ml ethidium bromide, which has been pre-equilibrated to cold room temperature. Add enough buffer to cover the gel (standard "submarine" conditions) and let the gel equilibrate to cold room temperature (at least 30 min if the buffer was pre-equilibrated; longer if not). While waiting for temperature equilibration, place the soon-to-be alkaline second dimension gel into its electrophoresis box and add enough alkaline electrophoresis buffer (AEB; 30-40 mM NaOH, 2 mM EDTA) to cover the gel. Incubate for at least one hour at room temperature (sufficient time for the gel to be permeated with AEB) before running the gel.
Before beginning electrophoresis, add appropriate markers to the marker wells. Size markers are especially important for the alkaline gel because they will be used to determine the minimum nascent strand length detectable with each probe. For the alkaline gel, the marker DNAs should be denatured by putting them in AEB for about 1 hour (take a few µl of the AEB out of the gel box and add it to your marker DNA). We have had good luck with the "1 kbp ladder" from BRL. If 100 ng of this marker are used in a single well, the bands are clearly visible by ethidium bromide fluorescence (even at 360 nm) in neutral gels, and, with random-oligonucleotide-primed probes synthesized from gel-purified restriction fragments, there is usually sufficient contaminating vector DNA in the "gel-purified" fragment that detectable signal is produced by the 1.6-kbp and 500-bp bands (derived from pBR322), providing useful reference points in every autoradiogram. Detection of the true "1 kbp ladder" (derived from yeast 2 µm plasmid) requires use of an intentionally radio-labeled 1 kbp or 2 µm probe. Dye markers are also useful during second dimension electrophoresis. Standard dyes can be used in the neutral gel. For the alkaline gel use 0.1% bromcresol green. Bromcresol green is more stable under alkaline conditions than bromphenol blue, xylene cyanol, or orange G. Its electrophoretic properties are indistinguishable from those of bromphenol blue. Remember to adjust the ionic conditions of your marker samples so they match those of the corresponding electrophoresis buffers. Also remember to add Ficoll.
Neutral electrophoresis is carried out at relatively high voltage. We have used two conditions successfully: 125 V (for a 20 cm gel)(starting current should be about 125-150 mA) for about 3 hrs or 80 V for 7-12 hours. The first conditions give satisfactory results in a shorter time; the second conditions give somewhat higher resolution, and may give clearer results for larger restriction fragments (6-15 kbp), but in a longer time. It seems likely that all conditions intermediate between these two sets of tested conditions would also work. Furthermore, additional conditions beyond the range of these tested conditions may also work.
Because the neutral gel contains ethidium bromide, progress of the run may be monitored with UV light. Be sure to use only 360 nm light for monitoring because shorter wave lengths may introduce nicks which could lead to electrophoretic abnormalities on resumption of electrophoresis. Good results are obtained when a 500 bp marker has migrated about 8 cm from the origin. Note: the reason we use a 20 X 20 cm gel format is not because we need the full 20 cm length (we don't), but because we need the full 20 cm width to accommodate two first dimension gels. In fact, one should not carry out electrophoresis for longer than necessary or the signal from replicating molecules will be diluted by being spread out over too large a portion of the final gel.
When neutral electrophoresis has been completed, the gel is photographed (any wavelength is acceptable; 300 nm is recommended) to visualize the markers and the arc of linear double-stranded restriction fragments. Replication-fork-containing restriction fragments form arcs, visible only by use of radiolabeled probes, above the arc of double-stranded linear molecules. In some cases, additional arcs may be visible by fluorescence: single-stranded DNA (faintly visible only in caffeine-wash fractions) forms an arc which rises above the double-stranded arc, all the way to the top of the gel. RNA (which should not be present, but occasionally is anyway) forms an arc below the double-stranded arc in the low-molecular-weight region of the gel. The major, double-stranded arc should be easily visible. If it is faint or absent, there is probably not enough DNA in the gel to detect signals from replicating unique-sequence yeast DNA. After the neutral gel has been photographed it can be wrapped in Saran wrap and stored in a refrigerator or cold room until the alkaline gel has finished, or it can be blotted to a nylon membrane right away. Although we have not tested this possibility, a report in BioTechniques (Vol 14, June 1993, pages 932-935) raises the possibility that large amounts of ethidium bromide in the gel may interfere with subsequent hybridization. Therefore, it may be a good idea to de-stain the neutral gel after it has been photographed and before it is blotted to a nylon membrane.
Alkaline gel electrophoresis is carried out at low voltage overnight. This is largely because the conductivity of AEB is high, forcing the use of low voltages to prevent excessive heating and distortion of the gel. Good results are obtained using 15 V (20 X 20 cm gel) for 17 hours (starting current should be about 60-70 mA), but voltage and time can be altered over a considerable range without impairing the quality of the results. If previously untested conditions are being used, then a good criterion for completion of electrophoresis is migration of the bromcresol green (BCG) marker; it should travel about 7 cm for good resolution of single strands in the range from 200 bp to 5 kb. It's difficult to resolve strands larger than about 8 kb in a 1% agarose gel; fortunately, it's not usually necessary to do so. Note that the BCG dye usually diffuses to invisibility in an overnight run. If one intends to rely on it as a marker, a second aliquot of BCG-containing sample solution (with AEB and Ficoll but without marker DNA) should be added to the marker wells when the run is about half over, and the distance between the old and new BCG spots should be noted. Later on, after the old BCG has disappeared, the position it would have if it were visible can be calculated simply by adding the distance measured in the previous sentence to the current position of the second BCG spot. Why don't we use ethidium bromide fluorescence to tell when the alkaline electrophoresis is completed? Ethidium bromide is not a sensitive detector of single-stranded DNA; to our knowledge there is no sensitive fluorescent detector of single-stranded DNA which works under alkaline conditions.
It is advisable to treat the gels briefly with acid in order to partially (only slightly) depurinate the DNA. Upon subsequent transfer to alkaline conditions, each apurinic site will be cleaved. The goal is to introduce apurinic sites at a frequency of 1 per 500-1000 bases so that most strands will be < 1 kb in length during alkaline transfer. Strands longer than 1 kb may not be efficiently transferred.
Soak large (20 X 20 cm) gels in at least 500 ml each of 0.25 M HCl. The gels should be rocked gently on a shaker while soaking, if possible. Soak the gels for 10-15 min if they are thin (< 5 mm thick) or 20-30 min if they are thick (up to 1 cm thick). The bromphenol blue or bromcresol green dye should turn yellow during the acid soaking. Do not soak longer than recommended. Our results show that prolongation of the depurination time to 40 min leads to significant decrease in signal. After completion of soaking, rinse the gels with distilled water and then begin alkaline transfer as quickly as possible.
Our tests indicate that the optimum buffer for alkaline transfer (either capillary or vacuum) is 0.4 M NaOH, 1 M NaCl. For most nylon membranes (see next paragraph), the NaCl is very important for effective transfer of strands longer than 1 kb, and, despite the depurination procedure, some strands may remain longer than 1 kb. In some experiments, vacuum transfer has given somewhat better results than those obtained with capillary transfer. However, in other experiments results obtained with vacuum transfer have been terrible. We do not yet know the reasons for these variations and consequently recommend capillary transfer at present.
We have tested several transfer membranes from different manufacturers and found significant differences in the final signal after hybridization. We don't make any specific recommendations here, because we have also noticed that the properties of membranes from any single manufacturer change with time. We simply recommend that the reader carry out similar comparison tests and use the type of membrane that gives the best results.
In our hands, standard capillary (Southern) transfer methods as described in standard laboratory manuals all work fine. Here we mention a few points which experience has shown to be important. Always wear clean gloves when handling membranes. Cut the nylon membrane to the size of the gel or slightly smaller. While the membrane is dry, mark it with a water-resistant marker (along the edge) with important information such as date and type of material being transferred. The markings should indicate, either directly or indirectly, which side of the membrane will contain DNA and which side of the membrane corresponds to the top, left, right, and bottom of the gel. Handle the gel carefully; it's best to use a supporting piece of plastic or glass while placing the gel onto the wick surface. Make sure there are no air bubbles between wick and gel, then pour additional transfer buffer onto the top surface of the gel and carefully apply the nylon membrane to that surface.
Each layer of the "transfer pyramid" should be cut slightly smaller than the previous layer to prevent channeling of fluid flow in the wrong direction (a frequent cause of poor transfers). Overlay the membrane with one or two pieces of wet filter paper (GB002 is fine) cut slightly smaller than the membrane, then add a stack (1-2 cm) of dry filters (same size) followed by a stack (2-3 cm) of paper towels cut slightly smaller than the filter papers. Place a flat piece of glass or plastic on top of the paper towels and then place a weight (no more than 400 g for a 20 X 20 cm gel; proportionately less for smaller gels) on top of the plastic or glass to keep everything in contact. Too much weight decreases transfer efficiency due to gel collapse.
After transfer is completed, disassemble the "transfer pyramid". The membrane should be washed with a dilute neutralizing buffer such as 2 X SSC (3 washes, 5 min each) in order to remove alkali and salt, and then it should be air dried.
After drying, the membrane should be irradiated on its DNA surface with 254 nm UV light for the time experimentally determined in each laboratory to give optimum signal. This is important. Our tests have shown that the signal can be increased up to 5-fold with optimum UV irradiation, but if irradiation is carried out for too long, the signal can be significantly decreased or even eliminated! The UV exposure appears to covalently crosslink the DNA to the membrane, minimizing DNA loss during washing. However, if too many crosslinks are introduced, less DNA is available for hybridization.
We welcome all suggestions for improvement of these protocols. Please let us know.
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