Clinical Chemistry 43: 30-33, 1997;
(Clinical Chemistry. 1997;43:30-33.)
© 1997 American Association for Clinical Chemistry, Inc.
Optimization of single-strand conformation polymorphism analysis in the presence of polyethylene glycol
Arseni Markoff1,2,
Alex Savov2,
Vladimir Vladimirov1,
Nadia Bogdanova2,
Ivo Kremensky2 and
Varban Ganev1
1
Laboratory of Molecular Biology and Genetics, Department of Chemistry and Biochemistry, Medical University Sofia, 2 Zdrave Str., 1431 Sofia, Bulgaria.
2
Laboratory of Molecular Pathology, University
Hospital of Obstetrics and Gynaecology, 2 Zdrave Str., 1431 Sofia,
Bulgaria.
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Abstract
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We report optimization of single-strand conformation polymorphism (SSCP)
analysis in the presence of polyethylene glycol. The protocol developed
separates single-strand conformers in a much shorter time (1–3 h) than
conventional SSCP protocols and broadens the applicability of SSCP
analysis from 150 to as much as 500 bp of DNA by different percentages
of GC content present. We conclude that addition of polyethylene glycol
helps improve the differential separation of conformers and, in
combination with high-resolution polyacrylamide gel electrophoresis,
offers an alternative to previous SSCP analysis protocols. This
protocol should be very useful for clinical applications in routine
detection of mutations as well as for research purposes.
Key Words: indexing terms: polymerase chain reaction • detection of mutations and polymorphisms • electrophoresis, polyacrylamide gel
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Introduction
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The method of single-strand conformation polymorphism (SSCP)
analysis is one of the most widely used methods for mutation detection
in current laboratory practice.1
Its simplicity and versatility, together with its comparatively high
rate of mutation detection, ~80% (1), make it a method
of choice for screening DNA fragments in many research and diagnostic
applications. Many modifications to the original protocol developed by
Orita et al. (2) include variables that affect the gel
matrix, e.g., percentage of acrylamide monomer, cross-linking ratio,
buffer systems, addition of neutral compounds to the gel, and
electrophoresis temperature. The most preferred gel characteristics for
successful differential separation of single-strand conformers in the
range of 200–300 bp, where the method should be most sensitive and
reliable, are 12% acrylamide (3) and cross-linking ratios
(%C) between 1 and 3 (4). Under these conditions,
so-called long-fiber gels are formed, large-pore matrices that are
sufficiently dense for successful electrophoretic separations but with
better flexibility than gels of other compositions. Protocols developed
so far rely on at least two temperature standard points for
electrophoresis with the gels, between 4 °C and 25 °C. Addition
of neutral compounds—e.g., glycerol, 50–150 mL/L—gives better
electrophoretic separation of conformers in some cases
(4).
Studies on protein separation by sodium dodecyl sulfate–polyacrylamide
gel electrophoresis (SDS-PAGE) in Laemmli-formulated conditions
(5) show that addition of water-soluble long-chain
polymers (LCPs) improves the electrophoretic resolution
(6)(7). Separation of proteins in a broad
molecular-mass range is enhanced, resolving smaller and larger proteins
on the same gel. Both viscosity and volume exclusion appear to take
part in the mechanisms by which water-soluble LCPs affect the migration
of proteins in SDS gels. SSCP analysis—yet another electrophoresis
technique—relies on "mild" denaturation of DNA molecules, such
that both strands are separated but both retain their most
energetically favored condition of secondary and tertiary structure. In
that regard, separation performance by SSCP analysis is somewhat
similar to that of SDS-PAGE of proteins: It depends on size and shape
but not on net charge.
The protocol we describe, in which polyethylene glycol (PEG) is added
to the gels, augments "classic" SSCP analysis in terms of
simplicity and versatility, allows for greater sample throughput, and
extends the boundaries of SSCP analysis into the 400–500 bp range by
different GC content present. We developed this protocol to solve some
technical difficulties arising from the application of traditional SSCP
analysis for mutation detection in DNA fragments longer than 400 bp,
which was unable to separate single-strand conformers even at different
gel compositions and temperatures.
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Materials and Methods
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Gel composition and buffers.
For the experiments
described, we applied two different gel formats in a discontinuous
electrophoresis system: polyacrylamide gels with added PEG, and gels
with no additive. To prepare 500 g/L PEG stock solution, we dissolved
50 g of PEG 6000 (Sigma, Deisenhofen, Germany) in 100 mL of
deionized water and added 0.01 g of sodium azide. Gel composition
was 12% acrylamide with bisacrylamide %C = 2.5 and 5 g/L PEG
final concentration. The gel buffer was 60 mmol/L formic acid adjusted
to pH 9.0 with Tris (up to 1 mol/L) (8). The trailing
buffers used were either Tris-glycine or Tris-alanine (0.5 mol/L Tris
and 50 mmol/L glycine or alanine), pH 8.3–8.6.
Sample preparation.
To test the protocol performance, we
investigated PCR products varying in GC% content and length, ranging
from 157 to 528 bp (Table 1
). We used 5 µL of PCR product (500–800 ng of DNA), either
undiluted or diluted with an equal volume of 10 mmol/L EDTA–1 g/L SDS
solution. The diluted samples were denatured with formamide dye (980
mL/L formamide, 10 mmol/L EDTA, 0.25 g/L xylene cyanol FF, and 0.25 g/L
bromphenol blue) in a total volume of 20 µL. The undiluted samples
were subjected to denaturation with various decreasing concentrations
of formamide dye, the lowest being 150 mL/L, thus reducing the total
sample volume to 6 µL. After denaturation in a boiling water bath for
3 min, the samples were chilled immediately on ice.
Electrophoresis.
For electrophoresis we used vertical
slab gel units, the MightySmall SE 250 (Hoefer Scientific Instruments,
San Francisco, CA) with gels of 80 x 70 x 0.75 mm, and the
SE 660 unit with gels of 140 x 240 x 0.75 mm. The gels were
run on constant power, 5 W for the SE 250 and 30 W for the SE 660, for
a period concomitant with the length of PCR product to be analyzed
(generally 1–3 h). Several different temperatures were used—0 °C,
4 °C, 18 °C, and room temperature—attained by using a cryostat
connected with a pump feed to the cooling coil of the unit. After
completion of the electrophoresis, the gels were silver-stained
according to Budowle et al. (9) and dried for
documentation.
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Results
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protocol development
Gel composition and buffers.
As Figs. 1
–3 show, adding PEG to the gel aided the electrophoretic
separation of single-strand conformers. Using the same SSCP protocol
but without any other polymer additive to the acrylamide/bisacrylamide
matrix failed to distinguish DNA amplicons that differed in primary
structure. Under standard SSCP conditions (no PEG additive), DNA
amplicons from individuals carrying the Val 458/Met 458 polymorphism in
exon 8 of the C1 inhibitor gene (C1INH) (10)
could not be separated into single-strand conformers; however, these
conformers clearly migrate differently when 5 g/L PEG is added to the
gel (Fig. 3
). The reasonable increase in gel viscosity when 5 g/L PEG is
included as a neutral additive allows for better separation on shorter
migration distances. The use of a discontinuous electrophoresis system
allows concentration of the single-stranded species in the samples into
a very small volume, thus additionally increasing the resolution of the
polyacrylamide/PEG gel. From these experiments it follows that PEG at 5
g/L is suitable for differential separation of single-stranded DNA
fragments in the range of 150–528 nucleotides in 12% acrylamide gels
with %C = 2.5.
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Figure 3. SSCP analysis on C1INH exon 8:
(left) no PEG additive; (right) 5 g/L PEG
additive.
Each lane shows PCR products from individuals heterozygous for the
Val458/Met 458 polymorphism.
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The two different trailing buffers used have similar effects on
separation but result in better separation patterns than does 0.52
mol/L Tris plus 0.28 mol/L boric acid, pH 9.0 (results not shown).
Initially we used 0.25 mol/L Tris plus 25 mmol/L glycine or alanine for
a trailing buffer; this gave satisfactory separation results but with
moderate to severe band distortion. A buffer of higher ionic strength
(0.5 mol/L Tris and 50 mmol/L glycine) gave better separation (compare
the right panel of Fig. 1
with the right panels of Figs. 2
and 3
).
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Figure 2. SSCP analysis of LDLR exon 12:
(left) no PEG additive; (right) 5 g/L PEG
additive.
PCR products are from (lane 1) noncarrier of Hinc
II polymorphism (-/-), (lane 2) individual
heterozygous for the Hinc II polymorphism (+/-), and
(lane 3) individual homozygous for the Hinc II
polymorphism (+/+).
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Sample preparation and electrophoretic conditions.
For
heat denaturation of the samples, 150 mL/L formamide was sufficient,
without prior dilution of the PCR product. The various temperatures
investigated made no difference in the electrophoretic behavior of the
separated single-strand conformers from different samples, so all
subsequent runs were performed at room temperature or in a cold room.
The modified protocol developed is summarized in Table 2
.
protocol evaluation
To test the performance of this protocol, we selected PCR products
of various lengths (157–528 bp) from: exons 13 and 12 of the LDL
receptor gene (LDLR), exon 11 of the putative oncogene
BRCA1, exon 8 of C1INH, and exons 10, 11, and 13A
of the CFTR gene (Table 1
). The DNA fragments were obtained
through amplification of genomic DNA from individuals whose genotypes
for the selected regions differed, as characterized in advance by
sequencing. Some of the alleles analyzed in this study are common
polymorphisms for the tested regions; others are disease-causing
mutations in the heterozygous state. For the LDLR gene, the
two polymorphisms in exons 12 and 13, creating restriction sites for
Hinc II and Ava II accordingly (11),
were tested by the modified protocol. The samples from homozygous (+/+)
and heterozygous (+/-) individuals and from noncarriers of the
mutation (-/-) were clearly distinguished (Fig. 2
, right). To test
the applicability of the method for SSCP of amplicons in the 400–500
bp range, we selected samples from subjects with either of two
different mutations in CFTR exon 13A (13).
Comparison with negative controls for these mutations shows that the
modified protocol was able to distinguish the carriers from the
negative controls (Fig. 1
, lanes 2 and 3).
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Discussion
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The results show that the optimized protocol has good performance
over a comparatively broad range of DNA fragment length and for GC%
content <50% (although one of the amplicons assayed has a GC% of
62.2; see Table 1
). The use of PEG as a neutral additive is simple and
straightforward. PEG does not chemically interact with the gel matrix,
but its large size allows it to remain trapped within the matrix
throughout the time of electrophoresis. Presumably, its presence
modulates the rate of migration of the DNA single-strand molecules in a
way that provides additional means to further control resolution. As
has been previously shown for protein molecules in SDS-PAGE,
incorporation of water-soluble LCPs into the gels provides yet another
means to extend the resolution performance of PAGE for proteins or
other biopolymers (6)(7). By including PEG as
an additive to acrylamide gels in SSCP analysis, it is possible to
separate PCR amplicons in a broad size range, from 150 to 500 bp. This
in turn extends the boundaries of SSCP analysis application and adds to
the uniformity of the analysis technique. Longer DNA fragments, which
are difficult to separate by conformation by traditional SSCP analysis
protocols, can be separated by this modified protocol. Using
silver-staining (9) allows completion of all separation
and visualization of mutations within 4–7 h. The results obtained with
this protocol support its further application for routine mutation
research and diagnostic screening.
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Acknowledgments
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We thank Bernd Dworniczak and Juergen Horst from the Institute of
Human Genetics in Muenster, Germany, for the careful reviewing of the
manuscript and helpful discussions and Stefan Kirov and Ani Horvath for
their technical assistance.
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Footnotes
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2 Current address and address for correspondence: Institut fuer
Humangenetik der WWU Muenster, Vesaliusweg 12–14, 48149 Muenster,
Germany. Fax (+49 251) 83 6995; e-mail markoff@uni-muenster.de;
hj292{at}Cleveland Freenet.Edu. 
1 Nonstandard abbreviations: LCP, long-chain polymer;
SSCP, single-strand conformation polymorphism; PAGE, polyacrylamide gel
electrophoresis; %C, cross-linking ratio; PEG, polyethylene glycol;
SDS, sodium dodecyl sulfate; and PCR, polymerase chain reaction.
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Abstract
Introduction
G mutation (2183AA