Short Communication

Optimization of Single-Stranded Conformation Polymorphism

(SSCP) Analysis for Screening for the Estrogen Receptor-� Gene

Polymorphism P325P

Clin Chem Lab Med 2001; 39(7):612–614 © 2001 by Walter de Gruyter · Berlin · New York

Darja Bitenc and Janja Marc

Faculty of Pharmacy of Ljubljana, Ljubljana, Slovenia

Since there are no theoretical models for single-

stranded conformation polymorphism (SSCP) analy-

sis, conditions for detecting specific mutation must be

found experimentally. Previously, a time-consuming

(22 hours) SSCP method was used for the detection of

polymorphism in codon 325 (CCC to CCG; P325P) in

exon 4 of estrogen receptor-� gene. The aim of our

work was to study different gel loading buffers, addi-

tives to polyacrylamide gel, voltages, running times

and temperatures of electrophoresis, in order to de-

velop a better and faster SSCP analysis for screening

of P325P polymorphism. Our results show that a low

ionic strength gel loading buffer and 10% addition of

glycerol to the 8% polyacrylamide gel (37:1) are essen-

tial for the good separation of mutated and wild-type

single stranded conformers of exon 4. The most suit-

able conditions for electrophoresis were 300 V, 5 h and

22 °C. We concluded that a much faster SSCP analysis

for sreening of P325P polymorphism of estrogen re-

ceptor-� gene was developed. Although our final re-

sult could be applied only to the detection of the de-

scribed genetic polymorphism, we hope that the

results of our study will be helpful to analysts using

SSCP analysis in their mutation screening programs.

Key words: Single-stranded conformation polymor-phism (SSCP); Estrogen receptor-α gene; Polymor-phism; Genes.

Abbreviations: dsDNA, double-stranded DNA; ERα, es-trogen receptor-α, LB, loading buffer; LIS, low ionicstrength loading buffer; SSCP, single-stranded confor-mation polymorphism; ssDNA, single-stranded DNA;CC is CCC/CCC genotype; CG is CCC/CCG genotype; GGis CCG/CCG genotype.

Estrogen receptor-α (ERα) gene is located on chromo-some 6q25.1. It has more than 140 kb in length and issplit into eight exons measuring 684, 191, 117, 336, 139,134, 184 and 4537 bp, respectively (1, 2). Until now, fourpolymorphisms of ERα gene have been confirmed:PvuII (T to C) and XbaI (A to G) in intron 1, BstUI (GCGto GCC; codon 87) in exon 1, and P325P (CCC to CCG;codon 325) polymorphism in exon 4 (3, 4). Polymor-phism P325P in codon 325 was found to be associatedwith breast cancer (4). In our previous study codon 325

sequence polymorphism was found to be associatedwith bone mineral density (BMD) of femoral neck inpostmenopausal women (5) and recently with calciumexcretion in urine (6), suggesting the involvement ofthis polymorphism in the development of osteoporo-sis.

Single-stranded conformation polymorphism (SSCP)analysis was used for the detection of P325P polymor-phism. SSCP analysis is based on the different elec-trophoretic mobility of wild-type and mutated single-stranded DNA (ssDNA), which depends not only on thesize of DNA fragment but also on its conformation. Un-der non-denaturing conditions single strands of DNAare folded to specific conformations that presumablydepend on internal base-pairing between short seg-ments of ssDNA. These conformations usually changeif only a single base is exchanged (7, 8). Until now therehas not been an adequate theoretical model, whichwould predict the three-dimensional ssDNA structureand the results of SSCP analysis. However, many para-meters were empirically found to affect the sensitivityof SSCP analysis: (i) gel matrix composition; (ii) size ofDNA fragment; (iii) buffer composition; (iv) gel addi-tives; (v) gel temperature during electrophoresis; (vi)DNA concentration; (vii) and G+C content of fragment(9). In our case the length of DNA fragment was 355 bp,with G+C content of 54.9%. The method described byJurada et al. was time-consuming (22 h), and extensivecooling at 4 °C was required (5). The aim of our workwas to reduce the running time of the SSCP analysispreviously used, and thus change some other parame-ters in order to achieve good separation of conformersanalyzing P325P polymorphism of ERα gene. For thisreason, the influences of loading buffer, glycerol, volt-age, temperature and running time of electrophoresison the results of SSCP analyses were studied system-atically.

DNA was isolated from periphal blood leukocytesobtained from patients with osteoporosis by Millersalting-out procedure (10). Exon 4 of ERα was ampli-fied by polymerase chain reaction (PCR), using 0.5units of Taq polymerase. The reaction mixture (25.0 µl)(50 mM KCl, 15 mM Tris-HCl, 1.5 mM MgCl2, 0.2 mMdNTPs, 0.4 µM of each amplification primer) was ini-tially denatured at 95 °C for 12 min, followed by 35 cy-cles of denaturation at 94 °C for 30 s, annealing at 62 °Cfor 30 s, and extension at 72 °C for 60 s. PCR was com-pleted by final incubation at 72 °C for 7 min. The lengthof the PCR product was 355 bp. Purity and concentra-tion of the amplified PCR product was confirmed by 2%

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Bitenc and Marc: SSCP analysis and ERα gene 613

agaraose gel electrophoresis with ethidium bromidestaining.

Three main steps are involved in the SSCP method:denaturation of double-stranded DNA (dsDNA) to ob-tain ssDNA, electrophoretic separation, and detection ofseparated ssDNA, in our case by Silver staining of gels.

For DNA denaturation, 2 µl of PCR product wereadded to 5 µl of common loading buffer (LB): for-mamide, 0.5 g/l bromophenol blue, 0.4 g/l xylenecyanol, 0.02 M EDTA, 0.8 g/l NaOH, or to 20 µl of lowionic strength loading buffer (LIS): 0.1 g/l bromophenolblue, 0.1 g/l xylene cyanol, 100 g/l saccharose, andmixed well. The mixture was heated at 97 °C for 3 min,immediately placed on ice and loaded to 8% polyacry-lamide gel (acrylamide/bisacrylamide = 37:1) with ourwithout 10% of glycerol.

Electrophoresis was conducted in 0.5 × TBE buffer(50 mM Tris-base, 40 mM boric acid, 0.5 mM diNa-EDTA (pH =5.3)) at different voltages (120, 300, 350, 400V) and running temperatures (4 °C, 22 °C).

After electrophoresis, the gels were Silver stainedaccording to Blum et al. (11).

At the evaluation of gel loading buffer influence, twodifferent buffers, LIS and LB, were used. Visibility ofbands and, consequently, the sensitivity of the methodwere higher using LIS buffer. Figure 1 shows the effi-ciencies of ssDNA formation by heat denaturation withLB and LIS solution. The buffer for loading samplesonto gel seems to be important for good SSCP analysisbecause it represents the medium in which denatura-

tion of dsDNA takes place. The quantity and stability ofssDNA conformers are crucial for a good SSCP analy-sis. LB buffer contains denaturing agents formamideand NaOH, which prevent renaturation of ssDNA afterheat denaturation (8, 9). LIS buffer is devoid of dena-turing reagents, but with low ionic strength the declinein the melting temperature of DNA could be achieved.Furthermore, the addition of a large volume of LISbuffer to PCR product causes sample dilution and, con-sequently, the probability of DNA renaturation afterheat denaturation is minimized (12).

Comparing SSCP results obtained with or without10% glycerol in polyacrylamide gel, our results showthat addition of glycerol was essential. As reported pre-viously, the presence or absence of glycerol, amongother parameters, has an important influence on band-ing profiles of ssDNA (13). Separation of DNA frag-ments was enhanced by glycerol addition because ofthe decreased pH value of buffer and gel. Lower pH ofthe TBE buffer is the result of interaction of glycerolwith borate in electrophoresis buffer. In general, the de-tection of mutations is often improved at low pH (13). Inthe case of exon 4 of ERα our results show that glycerolnot only increases the sensitivity of SSCP analysis butalso makes it feasible. The addition of glycerol was es-sential to distinguish between mutated and wild-typesingle-stranded conformers (Figure 2).

Fig. 1 The efficiency of ssDNA formation by heat denatura-tion of exon 4 of ERα with LB and LIS buffer. Electrophoresiswas carried out on a 8% polyacrylamide gel, 37:1 at 22 °C, at300 V for 5 h.

Fig. 2 SSCP analyses of exon 4 of ERα in polyacrylamide gelwith (gel 10) and without 10% glycerol (gel 23). Electrophore-sis was carried out on a 8% polyacrylamide gel, 37:1 at 22 °C,at 300 V for 5h. CC is CCC/CCC genotype, CG is CCC/CCG geno-type, GG is CCG/CCG genotype.

Tab. 1 Conditions for the electrophoresis.

Voltage (V) 120 300 300 350 400Time (h) 20 7 5 5 5

In order to reduce analysis time, SSCP analyses atdifferent voltages and running times were performed(Table 1). Higher voltages (350 V, 400 V) reduced run-ning times and diffusion of sample into gel. On theother hand, high gel temperature occuring at high elec-trophoretic voltages could change the structure andmobility of the conformers when gel temperature ap-proached the melting point of the ssDNA. Since elec-trophoresis was carried on water-jacketed gel platewith a circulating water bath, the gel temperature wasconstant and most probably did not exceed the meltingtemperature. Surprisingly, not only the running timesbut also the mode of band mobility was affected byhigher voltages. The band patterns changed at voltagesaround 350 V. At a voltage below 350 V both mutatedconformers (sense and anti-sense strands) in CCG/CCG(GG) homozygous had similar mobility and bands werecloser together (Figure 3, gel 16) or even fused into oneband (Figure 3, gel 10). Conformers of wild-type geno-type in CCC/CCC (CC) homozygotes were well sepa-rated in these conditions. At higher voltages (350 V andmore) conformers of mutated DNA (GG homozygous)separated, while both wild-type DNA conformers (CChomozygous) had similiar mobility (Figure 3, gel 11 and12). We could not explain these observations. In accor-dance with the results obtained, electrophoresis at 300 V for 5 h was chosen as most suitable.

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614 Bitenc and Marc: SSCP analysis and ERα gene

Afterwards, SSCP analyses conducted at 4 °C and22 °C were compared. The separation of bands at alower temparature was less complete, and discrimina-tion of the heterozygotes and homozygotes for the mu-tated gene was not possible (Figure 4).

We can conclude that LIS buffer for sample loading,addition of 10% of glycerol and electrophoresis at 300 Vand 22 °C for 5 h represent the most suitable conditionsfor screening for the P325P polymorphism of ERα gene.Although our final result applies only to the detection ofthe described polymorphism, we believe that the re-sults of our study will be helpful to analysts using theSSCP method in their mutation screening programs.

Acknowledgements

This work was supported by the grant J3-1356-0312-99 pro-vided by the Ministry of Science and Technology. We thank M.Sc. Sanja Jurada for access of unpublished data for this man-uscript.

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Received 18 January 2001, revised 10 May 2001, accepted 17 May 2001

Corresponding author: Janja Marc, University of Ljubljana,Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana, SloveniaTel: +38 6 1 47 69 500; Fax: +38 6 1 42 58 031E-mail: janja.marc@ffa.uni-lj.si

Fig. 3 SSCP analyses of exon 4 of ERα at different voltagesand running times of electrophoresis. Electrophoresis wascarried out on a 8% polyacrylamide gel, 37:1 at 22 °C. CC is

CCC/CC C genotype, CG is CCC/CCG genotype, GG isCCG/CCG genotype.

Fig. 4 SSCP analyses of exon 4 of ERα at 4 °C (gel 25) and22 °C (gel 11). Electrophoresis was carried out on a 8% poly-acrylamide gel, 37:1 at 300 V for 5 h. CC is CCC/CCC genotype,CG is CCC/CCG genotype, GG is CCG/CCG genotype.

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