5

Southern Blotting as a Diagnostic

Method
Bronwen M. Harvey and Pirkko Soundy

1. Introduction
Nucleic acid hybridization is a process in which complementary sin-
gle strands of nucleic acids combine to achieve a stable double-stranded
nucleic acid molecule. This action has been used to establish a molecular
or genetic relatedness among organisms and to characterize their genomes.
Furthermore, this technique is one of several diagnostic tools useful for
detecting a wide variety of conditions. Since the determination of the basic
principles of duplex formation and stability in the 1950s, many variations of
the hybridization techniques have been developed. Southern first transferred
DNA fragments from agarose, after electrophoretic separation, onto nitro-
cellulose (1). The technique is known as Southern blotting. Alwine et al (2)
described shortly afterward a similar Northern blotting technique in which
separated RNA strands are transferred from an agarose gel to a suitable solid
support. A logical extension of these blotting techniques has been the dot or
slot blot in which the sample is applied directly to the solid support without
prior size separation (3). Over the years, these techniques have been further
developed and modified extensively by many researchers across the world.
The application of these methods is as varied as the procedures used (e.g., to
determine the changes in the nutritional state of an environment, to establish
taxa genetically, to distinguish pathogenic from nonpathogenic viruses, to
analyze gene structure).
This chapter restricts itself to the application of Southern blotting to provide
information relating to genetic diseases. The DNA sample, which can include
blood, tissue biopsies, buccal scraps, amniotic fluid, cultured cells, and so
forth, is generally digested using a restriction endonuclease and then subjected
to electrophoresis in a horizontal agarose gel. After sufficient time has elapsed
to achieve adequate separation of the required fragments, the gel is soaked in
an alkali solution to achieve denaturation of the double-stranded nucleic acid,
then neutralized and prepared for transfer. Transfer to a nitrocellulose, polyvi-
nylidene (PVDF), or Nylon membrane is achieved by a process of blotting in
which buffer is drawn through the gel and the membrane. The fragments carried

From: Molecular Biomethods Handbook, 2nd Edition. 55

Edited by: J. M. Walker and R. Rapley © Humana Press, Totowa, NJ
56 B. Harvey and P. Soundy

with the buffer are retained on the surface of the membrane. The retention is
made more permanent through a fixation process. The blot can then be used in
a hybridization with labeled probes to identify the fragments of interest.
There are many variations on this basic theme (4,5). Those procedures
requiring the identification of fragments in excess of 10,000 bases advocate
the use of a depurination step to improve the efficiency of large-fragment
transfer. Methodologies using positively charged nylon membranes often
omit the neutralization step and advocate the use of alkaline transfer buffer,
which can also serve as a fixative. There are many ways to achieve the trans-
fer of DNA from the gel to the solid support. Southern’s original method (1)
describes the use of a capillary transfer procedure, and this remains the most
widely used technique by far because of its low cost and convenience, transfer
is often an overnight step (see Fig. 5.1.). If speed is a requirement, the transfer
process can be shortened by using specialized vacuum blotting apparatus or elec-
troblotting devices. Such techniques allow transfer in 30–60 min compared to
several hours for capillary transfer.
The membrane of choice is determined by the sensitivity required and the
detection method to be used. The quantity of sample has a significant effect on
both of these. The use of nitrocellulose usually results in low backgrounds and
is recommended when the level of target is high. Nitrocellulose membrane is
available in supported and unsupported forms, depending on the manufactur-
ing method employed; however, the handling characteristics of the latter can
be poor. Unsupported membranes are produced when the active substrate is
cast as a pure sheet. Because of their fragile nature, unsupported membranes
should be handled with care. Supported membranes are those for which the
active substrate is cast onto an inert “web” or support.
Nitrocellulose membrane can bind 80–125 µg nucleic acid/cm2, which is
significantly less than the binding capacity of 400–600 µg/cm2 for a nylon

Fig. 5.1. Diagrammatic representation of capillary transfer apparatus; suitable for the
transfer of DNA or RNA fragments separated by gel electrophoresis to a suitable Nylon
or nitrocellulose membrane for example. Hybond is available from GE Healthcare
Bio-Sciences
 Chapter 5 Southern Blotting as a Diagnositc Method 57

membrane. Its ability to bind small molecules (<400 nts) is also poor, and
transfer buffers must contain high salt concentrations to ensure efficient
nucleic acid binding. Nylon membranes are available in uncharged and posi-
tively charged supported forms. Charged nylon has a higher binding capacity
and is particularly useful when working with low-molecular-weight nucleic
acid. Binding to a nylon membrane is independent of the ionic strength of the
transfer buffer. However, backgrounds can be elevated. Where repeated use of
a membrane in hybridization assays is needed, the use of nylon membranes is
strongly advised. Nylon is also recommended for use with medium- or low-
abundance targets and is, in general, the membrane of choice when working
with nucleic acids. PVDF membranes behave similarly to uncharged nylon,
but because of its hydrophobic nature, use in nucleic acid blotting is limited.
Fixation bonds the target nucleic acid to the membrane. Suboptimal fixation
will lower sensitivity by reducing target concentration and is particularly harm-
ful if the blot is to be used more than once. The principal fixation methods of
heat and ultraviolet (UV) light can be used with all types of membrane. Heat
fixation is very reproducible but requires a vacuum oven for nitrocellulose. UV
crosslinking, performed using an UV crosslinker (constant energy setting),
is faster than heat. Alkali provides a third alternative method when charged
Nylon membranes are used.
Original methods describe the use of a DNA probe radioactively labelled
by random priming or nick translation with (32P)dCTP to detect specific
nucleic acid fragments immobilized on nitrocellulose membrane. Since then,
many different methods for labeling nucleic acid probes ranging from short
oligonucleotides to longer DNA or RNA fragments have been developed
(6,7). Nonradioactive labeling kits and reagents are also available, finding
favor in a number of niche areas (8). The role of the hybridization buffer is
to provide conditions that promote hybridization between the labeled probe
molecules and its complementary sequence immobilized on the membrane,
and to simultaneously limit hybridization between sequences that are not
perfectly matched (6) Table 5.1 lists factors affecting the hybridization rate
and stringency (4–6). Many different formulations of hybridization buffers
have been developed, containing inorganic salts and blocking agents such as
Denhardt’s solution (mixture of bovine serum albumin (BSA), Ficoll 400,
and polyvinyl pyrrolidone), denatured DNA from salmon sperm (or other

Table 5.1. Factors affecting hybridization rate and stringency.

Factor How hybridization is affected
Temperature High temperature increases hybridization stringency;
temperatures below Tm are recommended
(for RNA long probe, 10–15°C below Tm; for DNA long
probe, approx 25°C below Tm)
Ionic strength Optimal hybridization in the presence of 1.5 M Na+;
lowering ionic strength increases stringency.
Destabilizing agents For example, formamide and urea;
(or Tm modifiers) used to lower the effective hybridization temperature
Mismatched basepairs Mismatches lower hybridization rate
Duplex length Hybridization rate increases with increased probe length
Viscosity Increases rate of filter hybridization
58 B. Harvey and P. Soundy

species), and heparin (4–6). A short prehybridization step in hybridization

buffer is usually carried out to reduce nonspecific background hybridization
before adding the labeled probe. This is especially important in genomic
Southern blots that contain all genomic sequences on the membrane.
Hybridization with the probe is usually carried out for several hours to allow
hybridization between low-abundance sequences. Although various rapid-
hybridization buffers containing volume excluders are available to speed
up this step. After hybridization, the blot is washed to remove unhybridized
probe. The stringency of washing is usually controlled by stepwise reduc-
tions in the ionic strength of wash buffer and/or by temperature (4–6). The
replacement of the old plastic bag technology with specialized temperature-
controlled rotisserie devices for performing hybridization and washes has
resulted in more consistent results and safer handing of radioactivity. After
washing, the blot is subjected to autoradiography with X-ray film to visualize
the bound probe (9).

2. Southern Blotting in the Diagnosis of Human Disease

Southern blotting has been applied to the diagnosis of many human diseases
at the molecular level. These genetic diseases are caused by point mutations,
gene rearrangements, or the amplification of genes or specific sequences
within the genome. These methods have in common that restriction-digested
genomic DNA is size-separated in agarose gel electrophoresis, transferred
onto the membrane, and hybridized with gene-specific probes.
Restriction fragment length polymorphism (RFLP) analysis was one of
the early methods to diagnose point mutations implicated in genetic diseases.
This method was based on the observation that if a point mutation changes a
restriction fragment recognition sequence, it is possible to detect this change by
Southern blotting analysis in which the affected restriction enzyme is used to
cut genomic DNA before analysis. The change in the size of detected fragments
with a gene-specific probe signals the presence of mutation in the analyzed
gene. For example, this method has been applied to the diagnosis of hemophilia
A, which is the most common inherited bleeding disorder, affecting approx 1
in 5,000 males worldwide. Hemophilia A is an X-linked, recessively inherited
bleeding disorder that results from a deficiency of procoagulant factor VIII
(FVIII). Affected males suffer from joint and muscle bleeds and easy bruising,
the severity of which is closely correlated with the level of activity of coagula-
tion factor VIII (FVIII:C) in their blood. Gitschier et al. demonstrated using
Southern blotting that it was possible to diagnose the disease in 42% of affected
families (10). Having identified the BclI polymorphism, X-linked inheritance
was demonstrated in three generations of a Utah family. DNA from a family
member was restricted with BclI, electrophoresed on a 0.8% agarose gel, and
blotted on to nylon membrane and probe with a complementary sequence
within the factor VIII gene labeled with radioactivity. Twelve bands were
observed by autoradiography. Eleven hybridizing bands remained constant, and
one varied in position at either approx 0.9 or 1.1 kb in size.
The presence of gene point mutations has also been diagnosed with Southern
blotting using allele-specific probes. These are usually short oligonucleotides in
which the mismatched sequence is situated in the middle. By carefully controlling
 Chapter 5 Southern Blotting as a Diagnositc Method 59

the hybridization and wash conditions (temperature and ionic strength of wash
buffers), it is possible to distinguish between the binding of oligonucleotides
differing by only one nucleotide. This method has been applied, for example,
to distinguish between the normal human β-globin gene and the sickle cell
β-globin gene (11,12). Sickle cell anemia is caused by a single base change,
resulting in the replacement of glutamic acid residue at position 6 of the pro-
tein (hemoglobin) by a valine residue.
Gene rearrangements can be diagnosed with Southern blotting if a probe
hybridizing to the affected areas is used. Rearrangement is detected by observ-
ing change in the size and pattern of hybridized genomic restriction fragments.
This type of analysis has been applied to the diagnosis of acute promyelocytic
leukemia (APL), a subtype of acute myeloid leukemia. The disease is charac-
terized by abnormal, heavily granulated promyelocytes, a form of white blood
cells. APL results in the accumulation of these atypical promyelocytes in the
bone marrow and peripheral blood, and they replace normal blood cells. At the
molecular level, the disease involves translocation between the retinoic acid
receptor-α (RAR-α) on chromosome 17 and the promyelocytic leukemia locus
(PML) on chromosome 15. This results in the transcription of novel fusion
messenger RNAs. By separating restriction-digested genomic DNA in pulse
field gel electrophoresis followed by hybridization with probes derived from
PML and RAR-α genes, it was possible to detect translocation events that cor-
related with disease progression (13).
Gene amplifications are implicated in many diseases. Charcot–Marie–Tooth
(CMT) syndrome is a common autosomal-dominant neuromuscular disorder.
The disease is characterized by a slowly progressive degeneration of the
muscles in the foot, lower leg, hand, and forearm and a mild loss of sensa-
tion in the limbs, fingers, and toes. The first sign of CMT is generally a high
arched foot or gait disturbances. Other symptoms of the disorder may include
foot bone abnormalities such as high arches and hammer toes, problems with
hand function and balance, occasional lower leg and forearm muscle cramp-
ing, loss of some normal reflexes, occasional partial sight and/or hearing
loss, and, in some patients, scoliosis (curvature of the spine). Genetically, the
disorder is usually characterized by duplication of a region on chromosome
17 through unequal crossover. As a result, affected patients carry three copies
of this region. One diagnostic approach to CMT, type 1A exploits Southern
blot hybridization and the relative intensity for three polymorphic MspI RFLP
bands within the duplicated area to judge whether patients have two or three
copies of this region using a region-specific probe. To normalize the observed
intensity of the signal resulting from the CMT gene probe, another probe
derived from unconnected sequences is used (14).
The most significant changes in the use of Southern blotting in diagno-
sis have been seen since the introduction of primer-specific polymerase
chain reaction (PCR) and automated nonradioactive sequencing techniques.
Mutation and gene deletions once detected via Southern blot analysis are
now routinely analyzed with these rapid and inexpensive methods, which are
often fully automated. Cystic fibrosis, Duchenne muscular dystrophy, sickle
cell anemia, and thalassaemia, to name a few, are now diagnosed by PCR.
PCR methods can be completed in as little as 4 h, whereas Southern blotting
can take up to 5 d to achieve the same result. More important, the amount
of DNA required for analysis is significantly less with PCR amplification
60 B. Harvey and P. Soundy

methods. The Southern blotting diagnosis method generally requires 5–10 µg

of genomic DNA. The introduction of a PCR-based method is generally only
achieved once the gene defect has been characterized at the molecular level. Hence
research into disorders where the defect is unknown or further information is
required still uses Southern blot analysis as an important research tool. In the
routine laboratory, the use of Southern blotting is restricted to those diseases
that require additional information the Southern blot can provide. One such
disease is Fragile X; however prescreening using PCR analysis is common.

2.1. Fragile X Syndrome

Fragile X syndrome is a common genetic disease. This inherited form of
mental retardation affects 1 in 4,000 males and 1 in 8,000 females (15). Males
with fragile X often exhibit characteristic physical features and accompanying
autistic and attention-deficit behaviors. Individual IQs are in the range 35–70
(16–18). Approximately 30% of females with full mutations are mentally
retarded, and their level of retardation is, on average, less severe than that
seen in males.
In 1943, Martin and Bell (19) were able to link the cognitive disorder to
an unidentified mode of X-linked inheritance. In 1967, Lubs (20) discovered
excessive genetic material extending beyond the low arm of the X chromo-
some in affected males. Diagnosis was originally based on cytogenetic analy-
sis of metaphase spreads, but less than 60% of the affected cells in affected
individuals showed a positive result. With this variability in the test, the carrier
status of individuals could not be determined. Interpretation of the result is
further complicated by the presence of other fragile sites in the same region
of the X chromosome.
The fragile X gene (FMR1) is located in chromosomal band Xq27.3 and
encodes an RNA-binding protein, which was initially characterized in 1991
(21–23) and contains a tandemly repeated trinucleotide sequence (CGG) end
at its 5' end. The disease is caused by the absence of a functional FMR1 gene
product (24). A small number of individuals classified as fragile X cytogen-
tically have expansion at the nearby FRAXE locus, which also contains an
unstable CGG repeat (25–28). The normal distribution of the repeat in the
unaffected population varies from 6 to 50. Affected individuals are classified
into one of two major groups; premutations of approx 50–200 repeats and full
mutations with more than 200 repeats. Some alleles with approx 45–55 copies
of the repeat are unstable and expand from generation to generation; others
are stably inherited (29–31). The larger the size of the female premutation, the
greater the risk of expansion during meiosis (32). Individual male or females
carrying a premutation are unaffected (20,29,30). Males pass on the mutation
relatively unchanged to all their daughters, all of whom are unaffected.
In some cases of premutation, an accurate estimate of the size of the expan-
sion is required, most especially in family studies. This increase in resolution
is achieved by Southern blot assay, using PstI digestion of genomic DNA
and detection with a probe close to the repeat array. Characteristic of the full
mutation is methylation of the promotor region of the gene (CpG island) (33);
this correlates directly to gene inactivation. This is an important event in the
disease pathogenesis, its effect on clinical severity is, however, unpredictable,
especially in females.
 Chapter 5 Southern Blotting as a Diagnositc Method 61

Today, two main approaches are used in diagnosis of fragile X syndrome:

PCR (36–39) and Southern blot analysis (40). The use of flanking primers
allows the amplification of the region of DNA containing the repeats. The size
of the PCR product is therefore indicative of the number of repeats present.
However, the efficiency of the PCR reactions inversely relates to the number
of repeats; hence, large mutations are more difficult to amplify and can fail
to yield a PCR product. False negatives by PCR can also be an issue caused
by the presence of normal and full mutations in some male patients (41). No
information as to the extent of methylation can be determined by a PCR-based
assay. Southern blotting allows both the size of the repeat segment and meth-
ylation status to be assayed simultaneously. Methylation- sensitive restriction
enzymes such as EagI or NruI can be used to distinguish between methylated
and unmethylated alleles. When combined with EcoRI, these enzymes give
fragment sizes of 2.6 kb from normal unmethylated FMR-I genes. Methylated
normal genes are not cut by these enzymes and yield 5.1-kb EcoRI fragments.
Methylated and unmethylated expansions are indicated by the presence of
bands or smears above the 5.1-kb and 2.6-kb fragments, respectively.
It is common practice in diagnosis laboratories to use PCR for prescreen-
ing and only to proceed to Southern blotting for those samples that fail to
amplify (males) or show a single normal allele (females). If the etiology of
mental impairment is unknown, DNA analysis for fragile X syndrome should
be performed as part of a comprehensive genetic evaluation, which includes
routine cytogenetic analysis. Cytogenetic studies are critical because consti-
tutional chromosome abnormalities have been identified as frequent or more
frequently than fragile X mutations in mentally retarded individuals referred
for fragile X testing. For individuals who are at risk because of an established
family history of fragile X syndrome, DNA testing alone is sufficient. If the
diagnosis of the affected relative was based on previous cytogenetic testing
for fragile X syndrome, then it is advised that at least one affected relative
should be included in the DNA testing profile. Prenatal testing (42) of a fetus
is indicated following a positive carrier test in the mother. When the mother
is a known carrier, DNA testing can be offered to determine whether the fetus
inherited the normal or mutant FMR1 gene. Results must be interpreted with
caution because the methylation status of the FMR1 gene is often not yet
established in chorionic villi at the time of sampling. Follow-up amniocentesis
might be necessary to resolve an ambiguous result. In a very small number of
patients, deletions or point mutations (43–48) rather than trinucleotide expan-
sion are responsible for the syndrome. In these cases, linkage (31,49,50) or
rare mutation studies are more useful.

References
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2. Alwine JC, Kemp DJ, Stark GR (1977) Method for detection of specific RNAs in
agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with
DNA probes. Proc Natl Acad Sci USA 74:5350–5354
3. Kafatos FC, Jones CW, Efstratiadis A (1979) Determination of nucleic acid
sequence homologies and relative concentrations by a dot hybridization procedure.
Nucleic Acid Res 7(6):1541–1552
4. Rapley R (ed) (2000) The nucleic acid protocols handbook. Humana, Totowa, NJ