* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download "Dot and Slot Blotting of DNA". In: Current Protocols in Molecular
DNA barcoding wikipedia , lookup
Membrane potential wikipedia , lookup
SNARE (protein) wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Molecular evolution wikipedia , lookup
Maurice Wilkins wikipedia , lookup
List of types of proteins wikipedia , lookup
Agarose gel electrophoresis wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Non-coding DNA wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Molecular cloning wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Endomembrane system wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Cell-penetrating peptide wikipedia , lookup
Transformation (genetics) wikipedia , lookup
Cell membrane wikipedia , lookup
DNA supercoil wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Nagamine, Y., Sentenac, A., and Fromageot, P. 1980. Selective blotting of restriction DNA fragments on nitrocellulose membranes at low salt concentrations. Nucl. Acids Res. 8:2453-2460. Noyes, B.E. and Stark, G.R. 1975. Nucleic acid hybridization using DNA covalently coupled to cellulose. Cell 5:301-310. Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517. Nygaard, A.P. and Hall, B.D. 1963. A method for the detection of RNA-DNA complexes. Biochem. Biophys. Res. Commun. 12:98-104. Stellwag, E.J. and Dahlberg, A.E. 1980. Electrophoretic transfer of DNA, RNA and protein onto diazobenzyloxymethyl (DBM)–paper. Nucl. Acids Res. 8:299-317. Peferoen, M., Huybrechts, R., and De Loof, A. 1982. Vacuum-blotting: A new simple and efficient transfer of proteins from sodium dodecyl sulfate–polyacrylamide gels to nitrocellulose. FEBS Letts. 145:369-372. Towbin, H., Staehelin, T., and Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applicatons. Proc. Natl. Acad. Sci. U.S.A. 76:4350-4354. Reed, K.C. and Mann, D.A. 1985. Rapid transfer of DNA from agarose gels to nylon membranes. Nucl. Acids Res. 13:7207-7221. Key Reference Seed, B. 1982. Diazotizable arylamine cellulose papers for the coupling and hybridization of nucleic acids. Nucl. Acids Res. 10:1799-1810. Smith, G.E. and Summers, M.D. 1980. The bidirectional transfer of DNA and RNA to nitrocellulose or diazobenzyloxymethyl paper. Anal. Biochem. 109:123-129. Smith, M.R., Devine, C.S., Cohn, S.M., and Lieberman, M.W. 1984. Quantitative electrophoretic UNIT 2.9B transfer of DNA from polyacrylamide or agarose gels to nitrocellulose. Anal. Biochem. 137:120124. Southern, 1975. See above. First description of capillary transfer from gel to membrane. Contributed by Terry Brown University of Manchester Institute of Science and Technology Manchester, United Kingdom Dot and Slot Blotting of DNA Dot and slot blotting are simple techniques for immobilizing bulk unfractionated DNA on a nitrocellulose or nylon membrane. Hybridization analysis (UNIT 2.10) can then be carried out to determine the relative abundance of target sequences in the blotted DNA preparations. Dot and slot blots differ only in the geometry of the blot, a series of spots giving a hybridization pattern that is amenable to analysis by densitometric scanning. Samples are usually applied to the membrane using a manifold attached to a suction device. The basic protocol describes such a procedure for dot or slot blotting on an uncharged nylon membrane; annotations to the steps detail the minor modifications that are needed if blotting onto nitrocellulose. The first alternate protocol describes the more major changes required for blotting with a positively charged nylon membrane. A second alternate protocol describes preparation of dot blots by spotting the samples onto the membrane by hand. CAUTION: In all of the protocols, wear gloves to protect your hands from the alkali solution and to protect the membrane from contamination. Avoid handling nitrocellulose and nylon membranes even with gloved hands—use clean blunt-ended forceps instead. Dot and Slot Blotting of DNA 2.9.15 Supplement 21 Current Protocols in Molecular Biology DOT AND SLOT BLOTTING OF DNA ONTO UNCHARGED NYLON AND NITROCELLULOSE MEMBRANES USING A MANIFOLD Dot and slot blots are usually prepared with the aid of a manifold and suction device. This is quicker and more reproducible than manual blotting and is the method of choice if a number of blots are to be prepared at any one time. Many commercial manifolds are available, most of them with interchangeable units that provide a choice of dot- and slot-blot geometries (Fig. 2.9.3). BASIC PROTOCOL In this protocol, the DNA to be transferred is heat-denatured and applied to the membrane in a salt buffer. After blotting, the membrane is treated with denaturation and neutralization solutions and the DNA immobilized by UV irradiation (for nylon) or baking (for nitrocellulose). Materials 6× and 20× SSC (APPENDIX 2) DNA samples to be analyzed Denaturation solution: 1.5 M NaCl/0.5 M NaOH (store at room temperature) Neutralization solution: 1 M NaCl/0.5 M Tris⋅Cl, pH 7.0 (store at room temperature) Uncharged nylon or nitrocellulose membrane (see Table 2.9.1, UNIT 2.9A, for suppliers) Whatman 3MM filter paper sheets Dot/slot blotting manifold (e.g., Bio-Rad Bio-Dot SF or Schleicher and Schuell Minifold II) UV-transparent plastic wrap (e.g., Saran Wrap) UV transilluminator (UNIT 2.5A) for nylon membranes 1. Cut a piece of nylon membrane to the size of the manifold. Pour 6× SSC to a depth of ∼0.5 cm in a glass dish; place the membrane on the surface and allow to submerge. Leave for 10 min. A nitrocellulose membrane should be wetted in 20× rather than 6× SSC. 2. Cut a piece of Whatman 3MM filter paper to the size of the manifold. Wet in 6× SSC. Use 20× SSC if transferring onto nitrocellulose. 3. Place the Whatman 3MM paper in the manifold and lay the membrane on top of it. Assemble the manifold according to the manufacturer’s instructions, ensuring that there are no air leaks in the assembly. 4. To each DNA sample, add 20× SSC and water to give a final concentration of 6× SSC in a volume of 200 to 400 µl. Denature the DNA by placing in a water bath or oven for 10 min at 100°C, then place in ice. dots slots Figure 2.9.3 Dot (left) and slot (right) blot manifold architectures. Preparation and Analysis of DNA 2.9.16 Current Protocols in Molecular Biology Supplement 21 The amount of DNA that should be blotted will depend on the relative abundance of the target sequence that will subsequently be sought by hybridization probing (see commentaries to UNITS 2.9A & 2.10). If using a nitrocellulose membrane, add an equal volume of 20× SSC to each sample after placing in ice. 5. Switch on the suction to the manifold device, apply 500 µl of 6× SSC to each well, and allow the SSC to filter through, leaving the suction on. For a nitrocellulose membrane, use 20× SSC. The suction should be adjusted so that 500 ìl of buffer takes ∼5 min to pass through the membrane, as higher suction may damage the membrane. Wells that are not being used can be blocked off by placing masking tape over them or by applying 500 ìl of 3% (w/v) gelatin to each one (the former method is preferable, as gelatin may lead to a background signal after hybridization). Alternatively, keep all wells open and apply 6× or 20× SSC instead of sample to the extra wells. 6. Spin the DNA samples in a microcentrifuge for 5 sec. Apply to the wells being careful to avoid touching the membrane with the pipet. Allow the samples to filter through. If any of the samples contain particulate material, blockage of the wells can be a major problem. If this occurs, add additional 6× SSC (20× SSC for nitrocellulose) and try to remove the blockage by resuspending the particles. The extra SSC must filter through the membrane, so often the blockage recurs. Increasing suction is not advisable, as it risks damaging the membrane. The best solution is to troubleshoot the DNA preparative method to avoid carryover of particles. 7. Dismantle the apparatus and place the membrane on a piece of Whatman 3MM paper soaked in denaturation solution. Leave for 10 min. 8. Transfer the membrane to a piece of Whatman 3MM paper soaked in neutralization solution. Leave for 5 min. 9. Place the membrane on a piece of dry Whatman 3MM paper and allow to dry. 10. Wrap the dry membrane in UV-transparent plastic wrap, place DNA-side-down on a UV transilluminator, and immobilize the DNA by irradiating for the appropriate time (determined as described in UNIT 2.9A support protocol). CAUTION: Exposure to UV irradiation is harmful to the eyes and skin. Wear suitable eye protection and avoid exposure of bare skin. UV irradiation causes DNA to become covalently bound to the nylon membrane. The membrane must be completely dry before UV crosslinking; check the manufacturer’s recommendations. A common procedure is to bake for 30 min at 80°C prior to irradiation. Plastic wrap is used to protect the membrane during irradiation, but it must be UV transparent. A UV light box (e.g., Stratagene Stratalinker) can be used instead of a transilluminator (follow manufacturer’s instructions). Nitrocellulose membranes should not be UV irradiated. Instead, place between two sheets of Whatman 3 MM paper and bake under vacuum for 2 hr at 80°C. 11. Store the membrane dry between sheets of Whatman 3MM filter paper for several months at room temperature. For long-term storage, place the membrane in a desiccator at room temperature or 4°C. Dot and Slot Blotting of DNA 2.9.17 Supplement 21 Current Protocols in Molecular Biology DOT AND SLOT BLOTTING OF DNA ONTO A POSITIVELY CHARGED NYLON MEMBRANE USING A MANIFOLD Positively charged nylon membranes bind DNA covalently at high pH (see UNIT 2.9A). Samples for dot or slot blotting can therefore be applied in an alkaline buffer, which promotes both denaturation of the DNA and binding to the membrane. The procedure is therefore quicker than blotting in salt buffer, as the post-blotting denaturation, neutralization, and immobilization steps are omitted. ALTERNATE PROTOCOL Additional Materials Positively charged nylon membrane (see Table 2.9.1, UNIT 2.9A, for suppliers) 0.4 M and 1 M NaOH (APPENDIX 2) 200 mM EDTA, pH 8.2 (APPENDIX 2) 2× SSC (APPENDIX 2) 1. Cut a piece of positively charged nylon membrane to the appropriate size. Pour distilled water to a depth of ∼0.5 cm in a glass dish; place the membrane on the surface and allow to submerge. Leave for 10 min. 2. Prepare the blotting manifold as described in steps 2 and 3 of the basic protocol, using distilled water instead of SSC. 3. Add 1 M NaOH and 200 mM EDTA, pH 8.2, to each sample to give a final concentration of 0.4 M NaOH/10 mM EDTA. Heat for 10 min in a water bath or oven at 100°C. Microcentrifuge each tube for 5 sec. The alkali/heat treatment denatures the DNA. The amount of DNA that should be blotted will depend on the relative abundance of the target sequence that will subsequently be sought by hybridization probing (see commentaries to UNITS 2.9A & 2.10). 4. Apply the samples to the membrane as described in steps 5 and 6 of the basic protocol, but prewash the membrane with 500 µl distilled water per well. 5. After applying the samples, rinse each well with 500 µl of 0.4 M NaOH and dismantle the manifold. 6. Rinse the membrane briefly in 2× SSC and air dry. 7. Store membrane as described in step 11 of the basic protocol. MANUAL PREPARATION OF A DNA DOT BLOT Dot blots can also be set up by hand simply by spotting small aliquots of each sample on to the membrane and waiting for the blot to dry. Repeated applications enable a sufficiently large volume of a dilute DNA sample to be blotted, but applying more than 30 µl is tedious and leads to untidy dots. In fact, manual application rarely produces results of publishable quality. It should be used only if no manifold is available. The protocol below details the procedure used for uncharged nylon membranes; annotations describe the changes needed to blot nitrocellulose and positively charged nylon membranes. ALTERNATE PROTOCOL CAUTION: Wear gloves to protect your hands from the alkali solution and to protect the membrane from contamination. Avoid handling nitrocellulose and nylon membranes even with gloved hands—use clean blunt-ended forceps instead. 1. Cut a strip of uncharged nylon membrane to the desired size and mark out a grid of 0.5-cm × 0.5-cm squares with a blunt pencil. Pour 6× SSC to a depth of ∼0.5 cm in a glass dish; place membrane on the surface and allow to submerge. Leave 10 min. A nitrocellulose membrane should be wetted in 20× instead of 6× SSC, and a positively Preparation and Analysis of DNA 2.9.18 Current Protocols in Molecular Biology Supplement 21 charged nylon membrane should be wetted in distilled water. 2. To each DNA sample, add 1⁄2 vol of 20× SSC to give a final concentration of 6× SSC in the minimum possible volume. Denature the DNA by placing in a water bath or oven for 10 min at 100°C, then place in ice. The amount of DNA that should be blotted will depend on the relative abundance of the target sequence that will subsequently be sought by hybridization probing (see commentaries to UNITS 2.9A & 2.10). The sample volume should be no more than 30 ìl and if possible much less. If necessary, reduce volume by ethanol precipitation (UNIT 2.1) before adding SSC. If using positively charged nylon, add 1 M NaOH and 200 mM EDTA, pH 8.2, to each sample to give a final concentration of 0.4 M NaOH/10 mM EDTA, then heat as described. If using a nitrocellulose membrane, add an equal volume of 20× SSC to each sample after placing on ice. 3. Place the wetted membrane over the top of an open plastic box so that the bulk of the membrane is freely suspended. 4. Spin each sample in a microcentrifuge for 5 sec, spot onto the membrane using a pipet, and allow to dry. Do not touch the membrane with the pipet when applying the samples. Up to 2 ìl can be spotted in one application. If the sample volume is >2 ìl, it should be applied in successive 2-ìl aliquots, with each spot being allowed to dry before the next aliquot is applied on top. Drying can be aided with a hair dryer, but be careful that the blower does not spread the sample over the surface of the membrane. Try to keep the diameter of each dot to <4 mm. 5. For an uncharged nylon or nitrocellulose membrane, denature, neutralize, and immobilize as described in steps 7 to 10 of the basic protocol. For a positively charged nylon membrane, rinse and dry as described in step 6 of the first alternate protocol. 6. Store membrane as described in step 11 of the basic protocol. COMMENTARY Background Information Dot or slot blotting followed by hybridization analysis (UNIT 2.10) was first developed by Kafatos et al. (1979). The procedure is used to determine the relative abundance of a target sequence in a series of DNA samples. If a manifold is used, a large number of samples can be applied at once, enabling many different DNAs to be screened in a single hybridization experiment. The technique has found many applications over the years. For instance, in genome analysis, information on the genetic significance of a DNA sequence can often be obtained by using the sequence as a hybridization probe to dot blots of DNA prepared from related species. The rationale is that most genes have homologs in related organisms; for example, a coding sequence from the human genome will probably hybridize to related sequences in dot blots prepared from DNA of various mammals. An intergenic or intronic region, which is less likely to have homologs in the other species, will probably not show widespread hybridization. This is the so-called “zoo blot” approach; blots containing DNA from a variety of related species are available ready-made from a number of suppliers. Critical Parameters The key requirement with dot blotting is that the DNA be fully denatured after transfer, or at least that all the samples be denatured to the same extent. The underlying assumption of dot blot analysis—that it can be used for meaningful comparisons of sequence abundance in different DNA samples—holds only if denaturation is precisely controlled. Variations in denaturation result in different samples having Dot and Slot Blotting of DNA 2.9.19 Supplement 21 Current Protocols in Molecular Biology different amounts of hybridizable DNA; if this occurs, the relative intensities displayed by two dots after hybridization will not be representative of the amount of target DNA that each contains. The protocols for blotting uncharged nylon and nitrocellulose membranes attempt to ensure complete denaturation through the use of two denaturation steps—a heat denaturation before application to the membrane and an alkaline denaturation after application. Heat denaturation on its own is rarely adequate, as the DNA can renature fairly extensively before application to the membrane, even if plunged into ice on removal from the incubator. Blotting, whether manual or with a manifold, takes time, with some samples being blotted more quickly than others, so differential renaturation is a possibility. The second denaturation step, when the membrane is placed on a filter paper soaked in alkali, is intended to bring all the DNA back to an equal standing. Note that these problems do not arise with alkaline blotting onto positively charged nylon, as the high pH of the blotting solution maintains the DNA in a denatured state. Alkaline blotting is therefore the method of choice for DNA dot and slot blots where comparisons between different samples are to be made. A second variable results from the purity of the DNA samples. With a Southern transfer, the gel electrophoresis step helps to fractionate away impurities, so the DNA that is transferred is relatively clean. Dot/slot blotting with bulk DNA lacks the benefit of a gel fractionation step, and the resulting co-blotted impurities can have unpredictable effects on hybridization, possibly reducing signal by blocking access to the hybridization sites, or increasing signal by trapping the probe. This must be taken into account if the signal intensity is to be used to estimate the absolute amount of target DNA, through comparison with a control dilution series. Copy number reconstruction by dot blot analysis is particularly suspect, as comparison between blots of cellular and plasmid DNA are reliable only if both types of DNA are scrupulously purified. problems with dot and slot blots become apparent only after hybridization analysis. The warning signs detailed in the commentary to UNIT 2.9A also hold for dot/slot blotting; other problems are described in UNIT 2.10 (see Table 2.10.4 for troubleshooting). Anticipated Results The procedures yield a clear white membrane carrying applied DNA in amounts up the carrying capacity of the matrix (Table 2.9.1, UNIT 2.9A). No data is generated until the membrane is subjected to autoradiography; anticipated results of autoradiography are discussed in UNIT 2.10. Time Considerations A manifold or manual blot can be set up and ready for sample application in as little as 15 min. After sample application, it takes about 3 hr to complete the protocol with a nitrocellulose membrane (most of this being the baking step), 60 min with an uncharged nylon membrane, and 30 min with a positively charged nylon membrane. The rate-determining step is sample application. Manifold application of clean samples (where no blockages occur) takes 5 min, but application by hand can take several hours if the sample volume is large and multiple additions have to made. Literature Cited Kafatos, F.C., Jones, C.W., and Efstratiadis, A. 1979. Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure. Nucl. Acids Res. 7:1541-1552. Key Reference Dyson, N.J. 1991. Immobilization of nucleic acids and hybridization analysis. In Essential Molecular Biology: A Practical Approach, Vol. 2 (T.A.Brown, ed.) pp. 111-156. IRL Press at Oxford University Press, Oxford. Describes dot and slot blotting in some detail. Contributed by Terry Brown University of Manchester Institute of Science and Technology Manchester, United Kingdom Troubleshooting As with Southern blotting (UNIT 2.9A), most Preparation and Analysis of DNA 2.9.20 Current Protocols in Molecular Biology Supplement 21