Clinical Chemistry 43:1 30 –33 (1997)
Molecular Pathology and Genetics
Optimization of single-strand conformation polymorphism analysis in the presence of polyethylene glycol Arseni Markoff,1* Alex Savov,2 Vladimir Vladimirov,1 Nadia Bogdanova,2 Ivo Kremensky,2 and Varban Ganev1
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 singlestrand 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
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. INDEXING TERMS: polymerase chain reaction • detection of mutations and polymorphisms • electrophoresis, polyacrylamide gel
The method of single-strand conformation polymorphism (SSCP) analysis is one of the most widely used methods for mutation detection in current laboratory practice.3 Its simplicity and versatility, together with its comparatively high rate of mutation detection, ;80% [1], make it a
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. *Current address and address for correspondence: Institut fuer Humangenetik der WWU Muenster, Vesaliusweg 12–14, 48149 Muenster, Germany. Fax (149 251) 83 6995; e-mail
[email protected]; hj292@Cleveland. Freenet.Edu. 3 Nonstandard abbreviations: LCP, long-chain polymer; SSCP, singlestrand conformation polymorphism; PAGE, polyacrylamide gel electrophoresis; %C, cross-linking ratio; PEG, polyethylene glycol; SDS, sodium dodecyl sulfate; and PCR, polymerase chain reaction. Received March 20, 1996; revised August 12, 1996; accepted August 22, 1996.
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Clinical Chemistry 43, No. 1, 1997
(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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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.
Results protocol development
Materials and Methods 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 5 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 Trisalanine (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 mL 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 mL. 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 mL. After denaturation in a boiling water bath for 3 min, the samples were chilled immediately on ice.
Gel composition and buffers. As Figs. 1–3 show, adding PEG to the gel aided the electrophoretic separation of singlestrand 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,
Fig. 1. SSCP analysis of CFTR exon 13A: (left) no PEG additive; (right) 5 g/L PEG additive. Shown are PCR products from (lane 1) individual heterozygous for the 2183AA3 G mutation (2183AA3 G/N), (lane 2) individual heterozygous for the 2176insC mutation (2176insC/N), and (lane 3) individual with wild-type genotype for CFTR exon 13A (N/N). Figs. 1–3: Analyses performed on 12% polyacrylamide gel with %C 5 2.5.
Electrophoresis. For electrophoresis we used vertical slab gel units, the MightySmall SE 250 (Hoefer Scientific Instruments, San Francisco, CA) with gels of 80 3 70 3 0.75 mm, and the SE 660 unit with gels of 140 3 240 3 0.75
Table 1. PCR products subjected to LCP-SSCP. Gene/exon
LDLR/13 LDLR/12 BRCA1/11 CFTR/11 C1INH/8 CFTR/10 CFTR/13A
Primers
Product length, bp
GC content, %
13a; 13b11 12a; 12b11 11K2.59; 11K2.3912 11i59; 11i3913 IN8A; IN81310 10i59; 10i3913 13i59; 13i3913
157 180 296 425 450 491 528
47.5 48.8 35.1 34.5 62.2 34.7 34.4
Fig. 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 (2/2), (lane 2) individual heterozygous for the Hinc II polymorphism (1/2), and (lane 3) individual homozygous for the Hinc II polymorphism (1/1).
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Markoff et al.: Optimization of single-strand conformation polymorphism analysis
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 5 2.5. 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). 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 singlestrand 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.
Fig. 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.
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 (1/1) and heterozygous (1/2) individuals and from noncarriers of the mutation (2/2) 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).
Discussion 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
Table 2. Optimized protocol for LCP-SSCP. Gel composition
Gel buffer
Trailing buffer
12% AA, C% 5 2.5%, 5 g/L PEG
60 mmol/L formic acid, pH 9.0 (1 mol/L Tris)
50 mmol/L glycine 0.5 mol/L Tris, pH 8.3
0.1 W/cm2 constant power t0 5 room temp. or 10 °C
Electrophoretic conditions
or 50 mmol/L alanine 0.5 mol/L Tris, pH 8.6
Running time 5 1–3 h
Clinical Chemistry 43, No. 1, 1997
conformation by traditional SSCP analysis protocols, can be separated by this modified protocol. Using silverstaining [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.
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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