Download "Dot and Slot Blotting of DNA". In: Current Protocols in Molecular

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
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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
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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
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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
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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
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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
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