Mol Biotechnol (2008) 38:155–163
DOI 10.1007/s12033-007-9006-7
REVIEW
PCR–SSCP: A Method for the Molecular Analysis of Genetic
Diseases
Kakavas V. Konstantinos Æ Plageras Panagiotis Æ
Vlachos T. Antonios Æ Papaioannou Agelos Æ
Noulas V. Argiris
Published online: 13 October 2007
Humana Press Inc. 2007
Abstract Single strand conformation polymorphism
(SSCP) is a reproducible, rapid and quite simple method
for the detection of deletions/insertions/rearrangements in
polymerase chain reaction amplified DNA. All the details
for the use of PCR–SSCP are presented in the direction of
genetic diseases (b-thalassaemia, cystic fibrosis), optimum
gel conditions, sensitivity and the latest modifications of
the method, which are applied in most laboratories. This
non-radioactive PCR–SSCP method can be reliably used to
identify mutations in patients (b-globin, CFTR), provided
suitable controls are available. Moreover, it is widely used
for mutation identification in carriers (b-thalassaemia,
cystic fibrosis), making it particularly useful in population
screening.
Keywords b-Thalassaemia Cystic fibrosis DNA
mutational analysis Single strand conformational
polymorphism Population characteristics
Abbreviations
ARMS
Amplification refractory mutation system
ASO
Allele specific oligonucleotide
Bi-ddF
Bi-directional dideoxy fingerprinting
Bp
Base pairs
CAE-SSCP Capillary array electrophoresis–SSCP
CE-SSCPC Capillary electrophoresis–SSCP
CF
Cystic fibrosis
K. V. Konstantinos (&) P. Panagiotis V. T. Antonios P. Agelos N. V. Argiris
Laboratory of Clinical Chemistry, School of Medical
Laboratories, Faculty of Health and Care, Highest
Technological Institute of Larissa, 37 Xenopulu str, Larissa
41221, Greece
e-mail: [email protected]
CFTR
DGGE
DHPLC
DNTP
EDTA
HD
IVS
PAGE
PCR
RFLP
SDS
SSCP
ssDNA or
dsDNA
TPE
TBE
TGGE
Cystic fibrosis transmembrane regulator
Denaturing gradient gel electrophoresis
Denaturing high-performance liquid
chromatography
Deoxynucleoside triphosphates
Ethylene diamine tetra-acetic acid
Heteroduplex
Intervening sequence
Polyacrilymide gel electrophoresis
Polymerase chain reaction
Restriction fragment length polymorphism
Sodium dodecyl sulphate
Single strand conformational polymorphism
Single stranded or double stranded DNA
Tris phosphate EDTA
Tris borate EDTA
Temperature gradient gel electrophoresis
Introduction
Identification of new genes improves our understanding not
only of single genes disorders, but also of complex disease
processes. Extensively described for the first time by Orita
[1] and Hongyo [2], SSCP analysis is used in many laboratories today. Several modifications and improvements
have been introduced to improve the differentiation of
conformational changes and migration of ssDNA. The
DNA fragments are either visualized by autoradiography of
labelled PCR fragments [3], silver staining [4], ethidium
bromide [5] or fluorescence labelled primers [6]. Assessments of b-globin and CFTR genes are to be used in this
review as examples of the role of SSCP within clinical
genetics.
156
Comparison with Other Techniques and SSCP
Modifications
Almost all methods of DNA analysis in haemoglobinopathies and genetic diseases currently in use are based on the
polymerase chain reaction [7]. Nowadays, numerous PCRbased techniques are employed in the detection of mutations [8]. All these techniques are discussed below.
Allele Specific Oligonucleotide Probes (ASO)
This method uses small synthetic DNA molecules, which
are composed in order to detect point mutations [9] and are
consisted of about 20 nucleotides. These oligonucleotide
probes undergo hybridization with fully complementary
sequences. The position of the modification is located in
the middle of the molecules. Usually, two probes are
synthesized per mutation under investigation. The first is
complementary to the normal sequence and the second is
complementary to the mutated-one. Then, the PCR product
is transferred in a hybridization nylon membrane and is
subjected to hybridization with the probes.
Restriction Fragment Length Polymorphism (RFLP)
Restriction enzymes have the ability to recognize and
cleave DNA at specific DNA sequences to generate a group
of smaller fragments. Frequently, a point mutation of a
gene leads to the elimination or generation of a recognition-cut point of the restriction enzyme [10]. For example,
in sickle-cell anaemia, change of an A base with a T base in
the gene of b-globin results in the substitution of the b6
aminoacid (valine instead of glutamic) and destroys the
recognition point of the restriction enzyme MstII (CCT-NAGG). In the aforementioned case, the point mutation is
easily recognizable after the multiplication of the part of
DNA, which includes the mutation and the incubation with
the proper restriction enzyme [11]. Then, the incubation
products are separated with agarose gel electrophoresis
using ethidium bromide as staining dye and are directly
developed with a UV radiation apparatus. The number and
the size of the DNA fragments are clearly affected by the
presence of mutation.
Amplification Refractory Mutation System (ARMS)
Briefly, for each DNA sample, four PCR reactions are set
up, containing a group of common primers, which function
as internal control for the PCR efficiency and a group of
two primers, one common and one specific, per mutant
Mol Biotechnol (2008) 38:155–163
sequence under investigation [12]. Appropriate controls,
containing known mutations or normal samples, are similarly amplified. The PCR products are analysed on a 2%
agarose gel for the presence of the appropriate size fragments, as previously described.
Single-Strand Conformation Polymorphism (SSCP)
Analysis
The SSCP process involves PCR amplification of the target
fragment, denaturation of the double-stranded PCR product
with heat and formamide (or other denaturants for example,
sodium hydroxide, urea and methylmercury hydroxide) and
electrophoresis on a non-denaturing polyacrilamide gel.
During electrophoresis ssDNA fragments fold into a threedimensional shape depending on their primary sequence.
The elecrophoretic mobility of separation affects the shape
of the folded, single stranded molecules. Even if the difference in the sequence between the wild-type sample
(Normal DNA N) and the examined fragment (Mutated M)
is just a single nucleotide, a unique electrophoretic
mobility will be adopted (Fig. 1).
The SSCP method is based on the observation that under
non-denaturing conditions, single stranded DNA (ssDNA)
fragments fell into unique conformations determined by
their primary sequence whose structures are stabilized by
intramolecular interactions [13]. As a consequence, even a
single base alteration can result in a conformational
change, which can be detected by the altered mobility of
the single-stranded DNA molecule in SSCP. Unfortunately,
no adequate theoretical model exists for predicting the
NORMAL DNA (N)
MUTATED DNA (M)
SINGLE STRAND DNA
N
M
Fig. 1 Concise presentation of SSCP method
Mol Biotechnol (2008) 38:155–163
three-dimensional structure assumed by a single stranded
DNA fragment of known nucleotide sequence under a
given set of conditions. Therefore, for each ssDNA fragment, the number of stable conformations, which give rise
to bands of different mobility during SSCP electrophoresis,
must be determined experimentally under rigorously controlled conditions.
Several parameters have been empirically found to
affect the sensitivity of SSCP analysis [14]. Among them
are (i) type of mutation; (ii) size of DNA fragment; (iii) G
and C content fragment; (iv) content of polyacrilimide or
other gel matrix composition; (v) gel size and potential;
(vi) gel temperature during electrophoresis; (vii) DNA
concentration; (viii) run time of the electrophoresis; (ix)
buffer composition, including ionic strength and pH; and
(x) buffer additives, such as glycerol or sucrose. All these
parameters are described in details by the authors in
Table 1 [6, 15–19].
Heteroduplex Analysis (HD)
The method is based on the formation of heteroduplexes by
mixing wild type and mutant DNA amplified by PCR [20].
The DNA fragment screened is subjected to denaturation
followed by reannealing. If a mutation is present in one of
the two alleles, four distinct species will be generated by
this reassortment: wild-type homoduplex (wt/wt), mutant
homoduplex (mt/mt) and two different heteroduplexes (wt/
mt). Different migration patterns of these re-formed double
strands are evident, depending on the presence or absence
of a mutation. The homoduplexes migrate faster through
the gel and are observed as intensely stained bands at the
bottom of the gel. On the other hand, the heteroduplexes
are delayed in the gel and are thus seen higher up and their
migration rate depending on structural characteristics. The
formation of heteroduplexes and their stability depends
primarily on the type of mutation in the fragment. Large
insertions or deletions ([3bp) create very stable heteroduplexes. This stability is insensitive to electrophoretic
conditions and permits easy detection of mutations.
Jackson et al. [21], also report that HA has specificity in
detecting microdeletions or insertions in the immediate
vicinity of the tested mutation site. However, heteroduplexes involving single-base substitutions are less stable
and oversensitive to environmental changes. Indeed, for
some single-base substitutions, the differences in mobility
between homo- and heteroduplexes are minimal or even
absent. This fact decreases the sensitivity of this method
for this type of mutations. Heteroduplex analysis is a
relatively new technique, which permits the rapid detection
of specific mutations, and has been successfully used in the
diagnosis of sickle-cell anaemia, cystic fibrosis [22], type
157
2B VWD, factor V Leiden, haemochromatosis and T-cell
lymphoproliferative disorders.
Denaturing Gradient Gel Electrophoresis (DGGE)
The electrophoretic separation of DNA molecules is only
based on the melting point of double stranded DNA [23].
The two complementary strands of DNA are separated
(melted) with the use of specific chemical denaturants
(formamide or urea).
Chemical Mismatch Cleavage Method (CMC)
This mutation detection method has been used successfully
in several genes. The principle of the CMC method is
based on the formation of heteroduplex double-stranded
DNA (dsDNA) by annealing single-stranded DNA
(ssDNA) from the wild-type and mutant alleles [24]. Those
points of heteroduplexes with non-complementary base
pairs are cut off with chemical reagents. The chemical
treatment of the heteroduplexes with hydroxylamine (H)
and osmium tetroxide (OT) cause chemical modifications
to the non-complementary C and T bases, respectively.
DNA is then cleaved at the modified base by piperidine,
and the products are separated by autoradiography or
fluorescence in polyacrylamide gel electrophoresis.
DNA Sequencing
With the use of DNA polymerase, the molecule of ds DNA
that is to be analysed, acts as template for the synthesis of
DNA copies that all have the same beginning but terminate
in different points of the strand [25]. The termination of the
reaction is fulfilled with the addition of dideoxyribonucleozites, which are unable to form 50 -30 -phosphodiesteric
bonds. The reaction is carried out fourfold, each time
adding one of the four dideoxyribonucleozites. The
resulting DNA molecules are separating with electrophoresis and the sequence is revealed by reading the gel
upwards. For their detection, the stable 50 -end of the double-stranded DNA is revealed with the use of fluorescent
dyes.
Simultaneous Performance of HA and SSCP
Heteroduplex analysis is most sensitive to insertions
and deletions, whereas SSCP is most useful for the
detection of point mutations. Therefore, a combined analysis offers increased sensitivity. During SSCP analysis,
CD41/42, CD47/48
214, 218, 322, 350
–
10
–
Vinyl polymer,
hydrolink-MDE,
phastsystem
8W
–
25 or 4
1:10
16
1· TBEa
Mutation
PCR size
GC or Tm of PCR
Polyacrylamide (%)
Cross linker (%)
Other gel matrix
Potential
Gel size (height · length · width) mm
Gel temperature (C)
Proportion or ng of DNA
Run time (h)
Buffer composition
–
Autoradiography
19
Detection limit (%)
Gel staining
References
b
Tris Phosphate EDTA
Tris Borate EDTA mmol/l
–
Sucrose (%)
a
0
Glycerol (%)
ADDITIVES
5 or 3
–
2
Introns
6
0 or 10
15
Silver stain
–
10–16mol per band
0 or 10
89a
14
0 or 10
133.5a
16
Silver stain
–
10
10
0.025 M Tris
0.088 M
L-glycine
1–2
44.5a
*5
15–62
100 · 100 · 3
300 V
–
–
12–20
Tm
2:5 or 50 ng
14
2.6
20
1,947, 326, 96,
154, 94, 93,
151, 148, 151,
118, 113, 153, 332
All gene
2
1, 2
2:22
20–24
140 · 160 · 1.5
–
Silver stain,
Fluorescence,
ethidium bromide
2.6
14
100–180 V
2.6
–
7.5
–
233
IVS-II-745
2
–
–
0 or 10
1:20
4
–
30 W
1, 2, 3.3
–
10
–
121, 408, 631
IVS-I-1, IVS-I-5,
CD17, CD26,
CD19, CD15
1
1, 2, 3
Exons
1
b-globin
Gene
Table 1 SSCP optimum gel conditions applied in b-globin and CFTR genes study
18
Autoradiography
82–90%
–
0
50 mM Tris borate,
4 mM EDTA
24
2:7
4 or room temperature
200 · 400 · 4
10 V/cm
5.2, 2.0
–
5, 7.25, 10, 9, 6
–
410, 491, 425,
485, 473, 386,
324, 236, 240
All gene
–
3, 4, 7, 10, 11,
12, 15, 21, 20
CFTR
41
Silver stain
–
–
0
1· TPEb
3
2:10 or *75 ng
4
180 · 200 · 5
10 W
2.0
–
8
–
98
DF508
–
10
158
Mol Biotechnol (2008) 38:155–163
Mol Biotechnol (2008) 38:155–163
159
double-stranded DNA molecules are usually not eliminated
completely. Furthermore, some of the denatured DNA (ss)
will inevitably reform into double strands (ds). Therefore,
there are numerous cases where the formation of heteroduplexes has been detected on gels used for SSCP
screening. Thus, the two methods can be simultaneously
performed on the same gel; fact, which increases the
detection sensitivity of both methods (estimated to more
than 95% for both missense and nonsense mutations). This
fact is of great importance for clinical diagnostic applications of both techniques.
The advantages and disadvantages of the methods
mentioned above are presented in Table 2.
The majority of these methods is used today in numerous laboratories worldwide and is discussed below. From
the above table, it is extracted that the most valuable
methods are DGGE, SSCP and sequencing. These methods
have the ability to detect either unknown mutations or two
mutations simultaneously. Besides, they are able to analyse
several haplotypes without the use of radioactivity and
under astringent conditions (for DGGE, SSCP).
The methods chosen for the diagnosis of the gene point
mutations or deletions, depend not only on the technical
expert personnel in the diagnostic DNA laboratory, but also
on the type and variety of the mutations likely to be
Table 2 Synoptic comparison of mutation detection methods
Method
Detection Easiness to establish
Use of
of two
radioactivity
mutations
Low Medium Maximum
ASO
No
*
Yes
RFLP
No
*
No
DGGE
Yes
*
No
SSCP
Yes
*
No
ARMS
No
Sequencing
Yes
*
No
Chemical
mismatch
No
*
Yes
Heteroduplex No
*
No
*
No
Table 3 Detection methods for known and unknown mutations
Known
Unknown
ASO
DGGE
RFLP
SSCP
ARMS
–
Sequencing
Chemical mismatch
–
Heteroduplex
encountered in the individuals (population groups) being
tested. In addition, the use of at least two alternative
methods for the detection of specific mutations has been
proved safer and more efficient.
For the identification of known mutations (Table 2) in
PCR-amplified DNA [26], the most commonly used procedures are the reverse dot blot analysis with allele specific
oligonucleotide probes (ASO) [27] and the amplification
refractory mutation system (ARMS) [28]. On the other
hand, unknown mutations can be detected with indirect
methods of denaturing gradient gel electrophoresis
(DGGE) [23] or single-strand conformation polymorphism
(SSCP) analysis [29], followed by direct sequencing. The
advantages and disadvantages of each of these methods are
discussed in the European Molecular Genetics Quality
Network [30]. Lately, systems based on a microelectronic
chip [31,32], denaturing high-performance liquid chromatography (dHPLC) [33], capillary electrophoresis CE–
SSCP [34,35], capillary array electrophoresis CAE–SSCP
[36,37], bi-directional dideoxy fingerprinting (Bi-ddF) [38]
and fluorescence-based PCR–SSCP [39] have been applied
for large scale, fast and reliable mutational screening of
known b-globin alleles. Besides, a promising method of
identification of unknown mutations is high-resolution
genotyping by amplicon melting analysis [40]. This is a
closed-tube system used for genotyping and mutation
scanning that does not require labelled oligonucleotides.
The factors, which are considered in the selection of a
particular mutation scanning technique are (i) the size of
the gene of interest; (ii) the available resource; (iii) the
degree of sensitivity and specificity required; and (iv) the
cost. Some laboratories are interested in large-scale
screening of a particular gene, spending time to optimize
conditions with DGGE or TGGE to achieve nearly 100%
sensitivity with a particular template. Once these conditions are standardized, then screening large numbers of
samples becomes routine. Other working groups may initially prefer SSCP for the mutations screening and
subsequently refinement under alternate condition or in
combination with heterodublex analysis to increase accuracy. The SSCP method can also be employed for the
identification and not simply the detection of the mutations
[41] (Table 3).
Specific Applications
Genes Responsible for Hereditary Diseases
PCR–SSCP has been used for the identification of mutations within candidate genes for many genetic disorders
[42–60]. For example, cystic fibrosis [CFTR] [61–63] and
b-globin [64] are two representative genes.
160
Mol Biotechnol (2008) 38:155–163
Polymorphisms
Identification of Known Mutations by SSCP
Even though RFLP analysis by Southern blotting technique
of the genomic DNA has been most frequently applied for
detection of polymorphisms, PCR-based methods are
generally recognized as superior for future applications
[65]. Various polymorphisms in cloned sequences have
been detected by PCR–SSCP including polymorphisms for
adenosine deaminase, angiogenin, serum albumin, a-acid,
glycoprotein, apolipoprotein, b-globin, 1a, HLA-DR-b, cmyc, low density lipoprotein receptor (1), interleukin [66],
dopamine D2 receptor [67], GLUT4 [68], insulinelike
growth factor 1 receptor [69] and thiamidine kinase [70].
This review summarizes for the first time the fact that the
SSCP method can be employed for the identification, and
not simply the detection, of the four most common bglobin gene mutations [IVS-I-110 (C to A): 43.2% (b+);
codon 39 (C to T): 20.9 % (bo); IVS-I-1 (G to A): 13.9 %
(bo) and IVS-I-6 (T to C): 8.8% (b+)] found in Greek bthalassaemic patients and carriers (Fig. 2a).
This was obtained after the comparison of the patterns of
their ssDNA electrophoretic separations obtained, with that
of appropriate controls. The result, however, was that each
genotype, and not each mutation, revealed a characteristic
pattern of bands. Thus, each of the four control heterozygous samples, showed a clearly distinct and individual
pattern of three or four bands (Fig. 2b).
Identification of known mutations using appropriate
controls has already been reported in the similarly indirect
detection method of DGGE. DGGE is thought to be of
greater sensitivity than SSCP and has the advantage that
the melting temperature of a nucleotide sequence can be
theoretically predicted. Therefore, the approach of diagnosing mutations in unknown mutated samples by
comparison with known controls can also be successfully
applied to SSCP; an intrinsically simpler method, because
the pattern of bands obtained depends on the precise
combination of alleles. This method stands for the
Additional Applications
PCR–SSCP has also been used to locate mutated sequences
in bacterial genes (Bacillus subtilis, gyrase A), hepatitis C
virus [71], human papiloma virus type 16 [72], searching
mitochondrial DNA point mutations [73]. Moreover, the
subtraction of mitochondrial cytochrom C oxidase subunit
I [74], identification of an enterovirus in aseptic meningitis
[75], the diagnosis of fungal infections [76] and the
investigation of ribosomal DNA [77] are some more significant examples of its contribution over understanding the
above cited diseases.
Fig. 2 (a) Diagrammatic
presentation of the 238bp
fragment of the b-globin gene
amplified with PCR. The
position of the four mutations
under study is indicated: 1.IVSI-1, 2.IVS-I-6, 3.IVS-I-110,
4.cd39. (b) Characteristic SSCP
electrophoretic analysis of bthalassaemic heterozygous
control samples. The samples
employed had the following
genotypes: Lanes: 1: IVS-I-1/
Normal, 2: IVS-I-6/Normal, 3:
Codon 39/Normal, 4: IVS-I110/Normal, 5: Normal control.
The box shows the bands due to
ssDNA
A
1 2
5′
3
4
3’
IVS-I-1
EXON 1
5 ′ Gl
B 1
3 ′ Gl
238 bp
238bp
2
IVS-II-1
EXON 2
3
4
5
EXON 3
Mol Biotechnol (2008) 38:155–163
determination of genotype of b-thalassaemic individuals,
which obviously is limited by the great number of controls
required. Loreover, the capability of SSCP to detect
mutations is lower compared to other methods, e.g. DGGE
or DHPLC, which are reported to detect more than 95% of
the present mutations [23]. Nevertheless, the SSCP method
is characteristically simple in application and success,
especially in b-thalassaemic carriers. The use of only four
heterozygous controls, each containing one of the four
most common mutations, could identify over 80% of the
carriers present in the Greek population. Thus an important
and useful initial screening of the target groups is built.
Moreover, this method could be successfully applied for
the identification, and not simply the detection, of mutations in other genetic diseases, especially if the number of
known mutations is not great as is DF508 mutation in
cystic fibrosis. Whereas SSCP could be employed for the
identification of CFTR gene DF508 mutation [18], it has
not been used for the determination of the percentage of
cystic fibrosis in Greece. Hereafter, this essay has additionally examined whether it can be used for the detection
and the identification of CFTR gene DF508 mutation
patients and carriers.
Conclusions
The PCR–SSCP is a novel and widely used method in
detection of mutations in both basic and applied biological
science. Thus, PCR–SSCP is considered still up-to date as
a method not only to screen potential sequence variations,
but also to identify new mutations. Moreover, its speed and
simplicity for detection of these/similar mutations make it
attractive for use in the clinical diagnostic laboratories.
Acknowledgements We thank Associate Professor S. Bonanou, for
many helpful suggestions and help in 2003 and Ms T. Tsianou for her
valuable contribution to the CFTR project. Grand sponsor European
Union—Archimedes II.
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