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CE U P D A T E — M O L E C U L A R B I O L O G Y I I
James Wisecarver, MD, PhD
Techniques Used To Test Native DNA
The first article of this series described the key
structural features of DNA and how it is extracted
from cells for study. After a DNA sequence has
been extracted from cells and purified, the genetic
information contained within the sequence can be
examined through a variety of techniques.
This article discusses the techniques commonly
used to test native high-molecular-weight DNA
obtained from cells and tissues, including
Southern blot, dot blot, and in situ hybridization.
Southern Blot
The technique known as Southern blot analysis is
used widely for analyzing the size of certain DNA
fragments. The sequence of interest is prepared
by digesting intact DNA collected from a patient's
cells with a restriction endonuclease enzyme.
Following complete digestion, a collection of
double-stranded DNA fragments remains, ranging in size from a few hundred to several thousand base pairs.
These double-stranded fragments can be separated by size using a technique known as agarose
gel electrophoresis. This technique involves placing the fragmented DNA into wells in a slab of
agarose gel and submersing the gel in a buffer
chamber in the electrophoresis apparatus.
Electric voltage is applied, causing the negatively
charged nucleic acid fragments to migrate toward
the positively charged region, or anode, of the gel
apparatus. The gel matrix acts as a sieve, allowing
the smaller fragments to move through the gel
matrix easily while the progressively larger fragments have more difficulty migrating through the
gel. These DNA fragments are invisible to the
naked eye but can be visualized if the gel is
stained with a dye such as ethidium bromide,
which binds to the DNA. After staining, the gel
can be placed on an ultraviolet light source.
Orange fluorescence indicates the presence of the
DNA within the lanes of the gel.
ABSTRACT Several techniques commonly are used to test
native high-molecular-weight DNA obtained from cells and
tissues, including the Southern blot, dot and slot blot procedures, and in situ hybridization of tissue sections with
oligonucleotide probes. These techniques work well when
there is an adequate amount of fresh tissue or cells available to
provide a source for the intact DNA. These techniques involve
separating, or denaturing, the double-stranded DNA into
individual strands, and then applying a marked nucleotide
probe and allowing it to hybridize to a complementary DNA
sequence that may be present. These techniques are used to
determine whether a particular DNA sequence is present.
This is the second article in a three-part series on DNA. Other articles discuss the structural properties of DNA, how it is extracted from cells for study, some of the basic tools
used to gain useful clinical information, and DNA amplification techniques. On completion of this series, readers will be able to describe the composition of DNA, how the
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fragments are prepared by enzyme digestion, separated using gel electrophoresis, and
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strands are arranged, how to extract DNA from tissues prior to testing, and how DNA
After the DNA fragments have been separated,
the double-stranded fragments are denatured
into single strands by soaking the gel in sodium
hydroxide followed by neutralization. These DNA
pieces then are transferred onto a piece of electrostatically charged paper or filter membrane. In
the original description of this technique, the slab
of gel was placed in a tray of buffer, and the filter
material was placed on top of the agarose gel.
Absorbent paper toweling was placed on top of
the filter material allowing the buffer to work
upward through the gel and the filter membrane
into the absorbent toweling (Fig 1). The migration of buffer also transfers the DNA fragments
from the gel onto the membrane surface. Most
laboratories use vacuum- or air pressure-blotting
devices that greatly shorten the time necessary to
transfer the DNA onto the membrane. The
single-stranded DNA fragments that have been
FEBRUARY 1997
VOLUME 28, NUMBER 2
From the
Department of
Pathology and
Microbiology,
University of
Nebraska Medical
Center, Omaha.
©
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0
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Reprint requests
to Dr Wisecarver,
Department of
Pathology and
Microbiology,
University of
Nebraska Medical
Center, 600 S 42nd
St, Omaha, NE
68198-3135.
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121
(
Test Your
Knowledge
Look for the CE
Update exam on
Molecular Biology
(702) in the March
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Fig 1. In the
Southern blot procedure, DNA fragments
that have been prepared by cutting
native DNA w i t h a
restriction enzyme
are separated in
agarose gel through
electrophoresis. The
fragments are transferred onto a nylon
membrane by placing the gel in a
buffer bath and placing the membrane
on top of the gel.
Paper toweling is
placed on top of the
membrane, drawing
the buffer upward
f r o m the sponge
located in the pan,
through the gel and
membrane, and into
the toweling. As this
occurs, the DNA
fragments leave the
gel and are deposited
on the membrane
surface, producing a
" b l o t . " After this
transfer is complete,
the fragments are
immobilized on the
membrane surface,
and a labeled probe
is added to localize
the DNA fragments
containing the
sequence of interest.
122
transferred are immobilized permanently onto
the membrane surface by brief exposure to ultraviolet light.
The next step is to determine the location and
size of the particular DNA sequence of interest.
An oligonucleotide probe with a base sequence
that is complementary to the sequence of interest
is prepared containing a label to permit detection.
One simple technique for labeling probes is to
incorporate radioactive nucleotides as the probe is
assembled. The location of the radioactive probe
can be detected by exposing the membrane to xray film. More recently, nonisotopic techniques
have been developed to label probes with enzymes
such as alkaline phosphatase. A suitable enzyme
substrate can be used to detect the presence and
location of the bound probe.
The membrane blot is placed in a salt solution
containing the labeled probe at a temperature
that will permit the probe to hybridize, or bind,
to its complementary DNA fragment bound to
the membrane. During the incubation period,
which often is several hours, the probe eventually
will find the complementary base pair sequence
on the membrane and bind to this target DNA.
Following hybridization, the solution containing
the labeled probe is removed, and the membrane
is washed several times with a salt-buffer solution
to remove any unbound probe from its surface.
By adjusting the salt concentration and the
temperature, one can carefully remove the nonspecifically bound probe, being careful not to disrupt the bonds between the probe and the
intended target sequence. This careful consideration of the ionic strength and temperature used
during the wash steps often is referred to as the
"stringency" of the procedure. If the temperature
is too low or the ionic strength is too high, the
probe might stick to the membrane in a nonspecific fashion. If the temperature is too high, the
probe might separate from the intended target
sequence. The appropriate conditions for each
procedure must be determined. When using a
commercially prepared kit, care should be taken
during probing and washing steps to adhere to
the times and temperatures outlined in the
instructions to obtain accurate results.
Following these wash steps, the membrane
should be exposed to x-ray film for a period of
hours to days, depending on the strength of the
signal emitted from the probe. If radioactive
probes are used, no further steps are needed.
When using enzyme-labeled probes such as alkaline phosphatase, however, a substrate must be
added to the membrane surface before exposure to
the film. This substrate, when cleaved by the alkaline phosphatase enzyme, emits photons of light.
These light photons act similarly to the particles
released from decay of a radioactively labeled
probe and alter the silver present in the x-ray film.
Development of the x-ray film following exposure
to the membrane surface yields an image that will
disclose the location of the DNA fragments containing the sequence of interest (Fig 2).
To determine the size of this fragment, a size
marker is placed in the gel and is transferred to
the membrane. By comparing the size of fragments
detected by the probe with the size marker, it is
possible to estimate the number of bases present in
the fragment to which the probe hybridized.
Most laboratories use a 1-kilobase DNA ladder in
which the sizing bands are approximately 1,000
bases apart.
Northern and Western Blots
The Southern blot was named for its inventor, Dr
Edwin Southern. Other investigators began to use
this technique to separate and identify RNA fragments and proteins. Investigators using this technique to isolate messenger RNA (mRNA) dubbed
it northern blotting, as opposed to Southern blotting, which examines DNA fragments. Not to be
outdone, protein chemists
used the term western
The Southern Blot Procedure
blotting to describe a technique in which proteins
are separated electroBuffer wicks up through
membrane into paper toweling
phoretically, transferred
Paper towels
Filter membrane
to membranes, and idenA
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tified through the use of
labeled antibodies specific
for the protein of interest.
Northern blot analysis of
mRNA is more technically
Gel containing
Buffer in tray
Sponge soaked in
demanding
than similar
separated DNA
buffer supporting gel
analyses of DNA. RNA is
fragments
LABORATORY MEDICINE
VOLUME 28. NUMBER 2
FEBRUARY 1997
degraded easily, because numerous
ribonuclease enzymes present in
mammalian cells quickly break
down these molecules. Investigators
performing such studies must be
careful to treat all reagents with a
substance such as diethylpyrocarbonate (DEP-C), which inactivates
these ribonucleases so that the RNA
sequence they are trying to detect is
not degraded. The Southern, northern, and western blot procedures
are similar in that large macromolecules are separated on a membrane,
followed by detection of a particular
sequence of interest or protein molecule using an oligonucleotide
probe or antibody marker system,
respectively.
Dot/Slot Blot Methods
Autoradiograph From Southern Blot
X-ray f i l m
__
Dark bands indicate
sites w h e r e the labeled
probe hybridized to the
DNA fragment being sought.
The size of the DNA fragments
can be estimated by comparing
them w i t h the DNA marker ladder
that w a s run on t h e same gel.
In Situ Hybridization
The previous sections describe techniques in
which DNA, removed from cells and tissues, is
separated and placed on artificial substrates and
then probed for sequences of interest. In some
cases, it is preferable to apply these labeled probes
directly to cells and tissues to localize the source
of the signal. This technique is known as in situ
hybridization. Using this method, the DNA within the native tissues is denatured by heating, followed by application of a labeled probe that
binds to the complementary sequence of interest
(Fig 4). Following several wash steps, a detection
method is used to localize the signal that indicates areas in which the probe has bound to the
tissues. In cases in which radioactive probes are
used, the tissue sections on microscope slides are
dipped in a silver emulsion similar to an x-ray
film. These slides are kept in the dark for a period
of time (from days to several weeks), after which
the emulsion is developed in a fashion similar to
developing film.
If you wish to determine whether a particular
nucleotide sequence is present in a patient's sample, shortcuts are available. In this analysis,
enzyme digestion and subsequent electrophoretic
separation need not be performed. Instead, the
DNA sample is rendered single-stranded by alkaline denaturation or heat, spotted onto a nylon
membrane, and then probed using an oligonucleotide probe that was labeled in the same way as
in the Southern blot technique. This procedure is
known as a "dot blot." For example, a probe specific for a particular mutation (eg, sickle cell anemia), can be used to test DNA from the patient's
cells to determine whether the mutation is present
(Fig 3). In this example, the probe is designed to
detect the thymine substitution for adenine,
which results in the incorporation of valine at
position 6 in the (3 hemoglobin chain. The
hybridization stringency conditions are adjusted
so that the probe will bind if
thymine is present, but will not bind
if adenine is present. This approach
The Dot Blot Technique
can detect individuals who have this
mutation owing to the presence of a
Patient DNA is spotted and linked to membrane substrate.
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Fig 2. An autoradiograph is prepared by
exposing x-ray film
to a membrane blot
that has been
hybridized with a
labeled probe. The
visible bands on the
film correspond to
the location where
the labeled probe
has become bound
(hybridized) to the
membrane. The traditional approach
has been to incorporate radioactive
nucleotides into the
probes. Recently,
nonradioactive
labels have been
developed that use
enzyme-labeled
probes and substrates that release
photons of light
when the enzyme
acts on it. These
light photons or
products of radioactive decay produce dark bands
on the film after it
is developed.
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Fig 3. The dot blot
technique involves
spotting DNA onto a
membrane substrate
and then using a
labeled complementary nucleic acid
probe to determine
whether a specific
sequence is present
in the specimen.
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123
Speaking the Language of DNA
This guide can help you better understand the techniques commonly
used to test native high-molecular-weight DNA.
Electrophoresis—A method for separating macromolecules on the
basis of size and net electrical charge.
Dot blot—A technique to determine whether a particular nucleotide
sequence is present in a patient's sample. A DNA sample is rendered
single-stranded by alkaline denaturation or heat, spotted onto a
nylon membrane, and then probed using an oligonucleotide probe.
Heterozygous—Possessing different copies of a gene from each parent.
Homozygous—Possessing identical copies of a gene from each parent.
Hybridization—Binding of two complementary base sequences.
In situ hybridization—A technique in which DNA within the native
tissues is denatured by heating, followed by application of a labeled
probe that binds to the complementary sequence of interest.
Oligonucleotide probe—A string of nucleotides used to detect the
presence of a complementary nucleic acid sequence.
Slot blot—The same procedure as dot blot (see above); the difference is the shape of the template used to spot the DNA onto the
membrane.
Southern blot—A technique to separate, identify, and determine the
size of DNA fragments.
Western blot—A technique in which proteins are separated electrophoretically, transferred to membranes, and identified through the
use of labeled antibodies specific for the protein of interest.
Alternatively, these probes can be labeled with
an enzyme similar to those used for blotting techniques. Following probe hybridization, the
enzyme substrate is placed on the slide, and a
color reaction develops at the site of probe localization. This process is similar to immunohistochemical detection of antigens, which is used
routinely in many pathology laboratories. In situ
techniques have been used for detecting viral
nucleic acid sequences (eg, human papilloFig 4. In the technique known as in
situ hybridization,
tissue sections are
mounted on slides,
and the DNA in the
section is denatured
by heating. A labeled
probe is hybridized
onto the section. The
presence of the
bound probe can be
detected by exposing the slides to a
film emulsion or by
adding the appropriate substrate if an
enzyme-labeled
probe is used.
124
In some cases, a fluorescent marker is attached
to the probe. These probes then are used in a
technique known as fluorescence in situ
hybridization (FISH) to detect sequences on individual chromosomes. Such techniques are useful
for detecting extra copies of a chromosome within cells (eg, trisomy 21 in Down syndrome).
Conclusion
DNA fragments that have been prepared by cutting DNA using bacterial restriction enzymes can
be transferred onto a solid supporting membrane
using Southern blotting. After the fragments have
been transferred to the membrane, a labeled
nucleic acid probe, having a sequence complementary to the target sequence, is used to pinpoint the location of the target sequence on the
membrane blot. Slot or dot blot techniques can
be used to detect the presence or absence of a target sequence within a mixture of unseparated
DNA pieces, without having to follow the steps of
electrophoretic separation.
In situ hybridization is used to identify a target
nucleic acid sequence within tissue sections. This
process is similar to immunohistochemistry techniques used in many laboratories. The difference
is that in situ hybridization incorporates a DNA
denaturation step to separate the cellular DNA
into individual strands. A labeled nucleic acid
probe then is used to detect the presence of the
target nucleic acid strand. In situ hybridization is
particularly useful for detecting viral nucleic
acids within tissues.®
Selected Readings
Darnell J, Lodish H, Baltimore D. Molecular Cell Biology.
New York, NY: Scientific American Books; 1990.
Kirby LT. DNA Fingerprinting: An Introduction. New York,
NY: Stockton Press; 1990.
Lewin B. The extraordinary power of DNA technology. In:
Lewin B, ed. Genes V. Oxford, England: University Press;
mavirus) within infected cells.
1994:633-656.
Piper MA, Unger ER.
Nucleic Acid Probes: A Primer
for Pathologists. Chicago, 111:
In Situ Hybridization
ASCP Press; 1989.
Ross DW. Tools of recombinant DNA technology. In:
Labeled probe hybridized
Ross DW, ed. Introduction to
to tissue
Molecular Medicine. New
Tissue section
York, NY: Springer-Verlag;
1992:29-52.
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