- Email: [email protected]
Improving the sensitivity of single-strand conformation polymorphism (SSCP) to study the variability of PLMVd
Journal of Virological Methods 135 (2006) 276–280
Short communication
Improving the sensitivity of single-strand conformation polymorphism (SSCP) to study the variability of PLMVd Wen Xing Xu a , Ni Hong a,c , Jian Kun Zhang a , Guo Ping Wang a,b,∗ a
College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China b Key Lab of Plant Pathology of Hubei Province, Wuhan, Hubei 430070, PR China c National Indoor Conservation Center of Virus-free Germplasms of Fruit Crops, Wuhan, Hubei 430070, PR China Received 4 December 2005; received in revised form 9 March 2006; accepted 16 March 2006 Available online 27 April 2006
Abstract Single-strand conformation polymorphism (SSCP) was used to characterize viroids. Eight cDNA clones, which showed identical profiles in preliminary existing SSCP analysis but had different sequences, were chosen to develop a sensitive SSCP technique for identifying the variability of Peach latent mosaic viroid (PLMVd). Polyacrylamide Gel Electrophoresis (PAGE) conditions were optimized to improve the sensitivity of the existing SSCP, and a modified SSCP protocol was developed. The results indicated that the modified SSCP protocol provided an overall sensitivity in identifying the variability of these clones, and showed higher resolution than the existing one and its improved versions. As shown by sequence analyses of cDNA clones of PLMVd and the modified SSCP profiles, there is no close correlation between the number of base changes and variation of the modified SSCP band patterns. The potential use of the modified SSCP analysis is discussed as a tool for viroids characterization. © 2006 Elsevier B.V. All rights reserved. Keywords: Peach latent mosaic viroid; SSCP
Viroids, the smallest infectious agents in plants, have singlestrand circular RNA genomes (245–401 nucleotides), and do not have a mRNA capacity and must recruit host proteins to assist in the steps of life cycle (Symons, 1997; Pelchat et al., 2003). Sequence variability has been reported in different viroid populations, and shows the high plasticity of these minimal genetic systems (Koltunow and Rezaian, 1988; Diener, 1996; Kofalvi et al., 1997). Many studies indicated that the change of a few nucleotides or even one nucleotide in viroid genomes could change dramatically their virulence to the host (Dickson et al., 1979; Visvader and Symons, 1985; Hammond, 1992; Reanwarakorn and Semancik, 1998; Qi and Ding, 2003). Therefore, it is necessary to develop a highly sensitive technique for understanding the relationship of molecular variation among viroids and their virulence. The single-strand conformation polymorphism (SSCP) technique is a feasible method for detection of point mutations and DNA polymorphisms (Orita et al., 1989a,b). Because of
∗
Corresponding author. Tel.: +86 27 87282590; fax: +86 27 87397735. E-mail address: [email protected] (G.P. Wang).
0166-0934/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2006.03.004
its speed, simplicity and applicability to large-scale screening, SSCP has been applied in plant virology to study molecular variability and molecular epidemiology, and to detect mixed infections (Rubio et al., 1996; Magome et al., 1999; Kong et al., 2000). SSCP has also been used to characterize Potato spindle tuber viroid, Citrus exocortis viroid and some other viroids, and the results have demonstrated that SSCP may be suitable for routine analysis of variability of their unknown sequences when the gel and electrophoresis conditions were optimized (Chen et al., 1995; Szychowski et al., 1998; Palacio and Duran-Vila, 1999; Foissac and Duran-Vila, 2000; Palacio-Bielsa et al., 2003). In this study, SSCP was used to study the genetic diversity of Peach latent mosaic viroid (PLMVd), a typical species of the genus Pelamoviroid, but the existing SSCP is not sensitive enough to distinguish small differences among different variants, even after changing several important parameters of the technique. A modified method is discussed based on the existing SSCP to study the variability of PLMVd. Young leaves of peach infected with viroids were collected from eight peach plants of the cultivar ‘Yuhualu’ with symptom of discoloration of peach latent mosaic disease in an orchard in spring, in the Hubei Province of China.
W.X. Xu et al. / Journal of Virological Methods 135 (2006) 276–280
Viroid cRNA was extracted according to the method of Zhang et al. (1998). The extracted viroid RNA was subjected directly to reverse transcription. The first-strand cDNA was synthesized using Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (Promega, Madison, WI) with primer1 (5 -ATCACACCCTCCTCGGAACCAA-3 ) comple-
277
mentary to the RNA sequence of PLMVd-P3 at 181–202 nt (Xu et al., 2005). Double-strand DNA was amplified with primer1 and primer2 (5 -CCAGGTAACGCCGTAGAAACTG3 ), which was homologous to the RNA sequence of PLMVd-P3 at 203–224 nt. PCR amplification was carried out using a thermal cycler (PTC-200; MJ Research Inc, USA) for 35 cycles of
Fig. 1. Alignment of sequences representing clone V1–V8.
278
W.X. Xu et al. / Journal of Virological Methods 135 (2006) 276–280
94 ◦ C/45 s and 60 ◦ C/45 s and 72 ◦ C/45 s after denaturation for 3 min in 94 ◦ C, and followed by a final extension of 7 min at 72 ◦ C. PCR products were subjected to electrophoresis in 1% agarose gel to confirm the synthesis of a DNA product of the expected size. The full-size PLMVd DNA of each sample was gel purified using agarose gel DNA purification kit (Takara Corporation, Dalian, PR China) and ligated to vector pMD18-T, followed by transformation into Escherichia coli DH5␣. The existing SSCP was carried out according to the method of Palacio and Duran-Vila (1999). Briefly, aliquots of 5 l of the RT-PCR products amplified from nucleic acid preparations were mixed with 8 l denaturing loading buffer (95% formamide, 10 mM NaOH, 0.05% bromphenol blue, 0.05% xylene cyanol), or aliquots of 0.2 l of the PCR products amplified from purified plasmids mixed with 2 l denaturing loading buffer, heated for 8 min at 100 ◦ C and chilled on ice for 3 min, then loaded into pre-run (200 V for 10 min) and pre-cooled (in 4 ◦ C for 30 min) 14% non-denaturing polyacrylamide (acrylamide:bisacrylamide, 49:1) gel (20 cm × 12.5 cm × 0.1 cm). Electrophoresis was carried out at 200 V in a refrigerator at 4 ◦ C in 1× TBE buffer (89 mM Tris, 89 mM boric acid, 2.5 mM EDTA, pH 8.3) for 2 h. The gel was then stained with silver according to the method of Igloi (1983). Using the existing SSCP system, eight samples of RT-PCR products amplified from nucleic acid preparations had eight identical profiles, as did eight clones (V1–V8) selected at random from these samples (data not shown). To understand the resolution of the existing SSCP, the eight recombinant plasmids were purified and sequenced with ABI PRISM DNA sequencer 377 (Perkin-Elmer). The sequences were analyzed using the Clustal V program (Thomson et al., 1994). The sequencing results indicated the eight clones showed changes from 1 to 25 nucleotides, including transitions, transversions, additions and deletions and covered most variable sites in the viroid and their homologies ranged from 99.7% to 92.4% (Fig. 1), which means the resolution of existing SSCP was very low. Some PAGE conditions were assayed to improve the resolution of the existing SSCP using the eight clones: (a) electrophoresis time (2 and 16 h); (b) gel concentration (14% and 8%); (c) running voltage (200 and 300 V); (d) cross-linking ratio of acrylamide and bisacrylamide (49:1 and 29:1). Based on these conditions, a modified SSCP was designed and the gel was prepared with two equal sections: the upper section was 8% polyacrylamide with a cross-linking ratio of 49:1, the bottom section was 8% polyacrylamide with a cross-linking ratio of 29:1. About 100 l isopropylalcohol was used to obtain a good horizontal cross-linked line of two parts of gel, and to cover the surface of the bottom part of gel before solidification and then removed after gel solidification, and the upper section gel was then prepared. Electrophoresis was carried out at 200 V for 16 h. Analysis of the profiles of electrophoresis indicated that prolonging the electrophoresis time from 2 to 16 h in 14% PAGE gel still had a low resolution. Among the eight clones analyzed, only clone V6–V8 showed different profiles with each other, but they could not be distinguished easily when they were mixed together, one clone remained undetected (Figs. 2A, 3A, 4A). When the gel concentration was reduced to 8% and other param-
Fig. 2. SSCP profiles of clone V1–V3 and mixed sample of the three clones in 14% PAGE gel with a cross-linking ratio of 49:1 at 200 V for 16 h (A). The same samples analyzed in 8% PAGE gel with a cross-linking ratio of 49:1 (B) and a cross-linking ratio of 29:1 (C) at 200 V for 16 h, and in 8% PAGE gel with a cross-linking ratio of 49:1 at 300 V (D), and in modified SSCP (E).
eters were unchanged, clone V1 could be discriminated with V2 and V3, but clone V2 and V3 showed same band migrations and still could not be discriminated from each other, and one clone remained undetected from mixed sample of V1–V3 (Fig. 2B). Mixed sample of V4 and V5 showed a three-band pattern, the upper two bands of them could not be differentiated (Fig. 3B). Under the same gel concentration, the changed voltage (300 V) and gel cross-linking ratio (29:1) could not give a higher resolution for mixed sample of V1–V3 (Fig. 2C and D), so did changed gel cross-linking ratio for mixed sample of V4 and V5 (Fig. 3C). In the modified SSCP analysis, the resolution was greatly improved, V1–V3 in single or mixed sample was clearly discriminated on the gel (Fig. 2E), so did V4 and V5 (Fig. 3D), V6–V8 (Fig. 4B). All eight clones examined showed different profiles and all the variations in these clones were dis-
W.X. Xu et al. / Journal of Virological Methods 135 (2006) 276–280
279
Fig. 4. SSCP profiles of clone V6–V8 and mixed sample of the three clones in 14% PAGE gel with a cross-linking ratio of 49:1 at 200 V for 16 h (A) and in modified SSCP (B).
Fig. 3. SSCP profiles of clone V4, V5 and mixed sample of the two clones in 14% PAGE gel with a cross-linking ratio of 49:1 at 200 V for 16 h (A). The same samples analyzed in 8% PAGE gel with a cross-linking ratio of 49:1 (B) and a cross-linking ratio of 29:1 (C), and in modified SSCP (D).
tinguished by the modified SSCP. Even a single base variation (T↔C in nucleotide 27) in variants V4 and V5 was clearly identified (Fig. 3D). No correlation existed between the number of base changes and variation of the SSCP band patterns, as reported previously by Palacio and Duran-Vila (1999). For example, total 12 base exchanges in V7 and V8 gave them more obviously distinct band migration than 17 base changes giving V2 and V3 in modified SSCP (lanes 2 and 3 of Figs. 2E and 4B).
The modified SSCP gave an overall sensitivity in identifying the variability of the eight clones and showed a higher resolution capability than the existing one and its improved versions. The modified SSCP with two cross-linking ratio of acrylamide and bisacrylamide within a gel summarized the resolution capacity of two conditions and gave an increased sensitivity. Clones such as V1 and V2 could not be distinguished in 8% gel with a crosslinking ratio of 29:1, but they could be distinguished in 8% gel with a cross-linking ratio of 49:1; clone V2 and V3 were on the contrary (Fig. 2B and C), when such two conditions were combined together, these samples could be distinguished in one test (Fig. 2E). Whether the modified SSCP could provide sufficient sensitivity to identify all existing sequence heterogeneity in complex mixtures of DNA samples needs further study. As a more sensitive variability identifying protocol the existing one, the modified SSCP analysis is not only used as a complementary tool in viroid characterization studies in which extensive sequencing is required, it could also provide valuable information for identification of molecular variability of viroid or RNA genomes, as well as for screening molecular variants, detection of mixed infections, comparison of field isolates. Acknowledgements This study was supported by National 863 Project of China (No. 2001AA241142). The authors would like to thank Profes-
280
W.X. Xu et al. / Journal of Virological Methods 135 (2006) 276–280
sor DaoHong Jiang, College of Plant Science and Technology, Huazhong Agricultural University, and Professor ShiYun Chen, Wuhan Institute of Virology, the Chinese Academy of Sciences for reading the manuscript and giving valuable suggestions. References Chen, X., Baumstark, T., Steger, G., Riesner, D., 1995. High resolution SSCP by optimization in the temperature by transverse TGGE. Nucleic Acids Res. 23, 4524–4525. Dickson, E., Robertson, H.D., Niblett, C.L., Horst, R.K., Zaitlin, M., 1979. Minor differences between nucleotide sequences of mild and severe strains of potato spindle tuber viroid. Nature 277, 60–62. Diener, T.O., 1996. Origin and evolution of viroids and viroid-like satellite RNAs. Virus Genes 11, 47–59. Foissac, X., Duran-Vila, N., 2000. Characterisation of two citrus apscaviroids isolated in Spain. Arch. Virol. 145, 1975–1983. Hammond, R.W., 1992. Analysis of the virulence modulating region of potato spindle tuber viroid (PSTVd) by site mutagenesis. Virology 187, 654– 662. Igloi, G.L., 1983. Silver stain for detection of nanogram amounts of tRNA following two-dimensional electrophoresis. Anal. Biochem. 134, 184–188. Kofalvi, S.A., Marcos, J.F., Ca˜nizares, M.C., Pall´as, V., Candresse, T., 1997. Hop stunt viroid (HSVd) sequence variants from Prunus species: evidence for recombination between HSVd isolates. J. Gen. Virol. 78, 3177–3186. Koltunow, A.M., Rezaian, M.A., 1988. Grapevine yellow speckle viroid: structural features of a new viroid group. Nucleic Acids Res. 16, 849–864. Kong, P., Rubio, L., Polek, M., Falk, B.W., 2000. Population structure and genetic diversity within California citrus tristeza virus (CTV) isolates. Virus Genes 21, 139–145. Magome, H., Yoshikawa, N., Takahashi, T., 1999. Single-strand conformation polymorphism analysis of apple stem grooving capillovirus sequence variants. Phytopathology 89, 136–140. Orita, M., Iwahana, H., Kanazawa, H., Hayashi, K., Sekiya, T., 1989a. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc. Natl. Acad. Sci. U.S.A. 86, 2766–2770.
Orita, M., Suzuki, Y., Sekiya, T., Hayashi, K., 1989b. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5, 874–879. Palacio, A., Duran-Vila, N., 1999. Single-strand conformation polymorphism (SSCP) analysis as a tool for viroid characterization. J. Virol. Meth. 77, 27–36. Palacio-Bielsa, A., Romero-Durban, J., Duran-Vila, N., 2003. Characterization of citrus HSVd isolates. Arch. Virol. 149, 537–552. Pelchat, M., Rocheleau, L., Perreault, J., Perreault, J.P., 2003. SubViral RNA: a database of the smallest known auto-replicable RNA species. Nucleic Acids Res. 31, 444–445. Qi, Y., Ding, B., 2003. Inhibition of cell growth and shoot development by a specific nucleotide sequence in a noncoding viroid RNA. Plant Cell 15, 1360–1374. Reanwarakorn, K., Semancik, J.S., 1998. Regulation of pathogenicity in hop stunt viroid related-group II. J. Gen. Virol. 79, 3163–3171. Rubio, L., Ayll´on, M.A., Guerri, J., Pappu, H., Niblett, C., Moreno, P., 1996. Differentiation of citrus tristeza clostervirus (CTV) isolates by singlestrand conformation polymorphism analysis of the coat protein gene. Ann. Appl. Biol. 19, 479–489. Symons, R.H., 1997. Plant pathogenic RNAs and RNA catalysis. Nucleic Acids Res. 25, 2683–2689. Szychowski, J.A., Credi, R., Reanwarakorn, K., Semancik, J.S., 1998. Population diversity in grapevine yellow speckle viroid-1 and the relationship to disease expression. Virology 248, 432–444. Thomson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Visvader, J.E., Symons, R.H., 1985. Eleven new sequence variants of citrus exocortis viroid and the correlation of sequence with pathogenicity. Nucleic Acids Res. 13, 2907–2920. Xu, W.-X., Wang, G.-P., He, Y.-W., Hong, N., 2005. The cloning and nucleotide sequence analysis of PLMVd isolate P3 from China. Acta Phytopathol. Sin. 35, 300–304. Zhang, Y.-P., Uyemoto, J.K., Kirkpatyick, B.C., 1998. A small-scale procedure for extracting nucleic acids from woody plants infected with various phytopathogens for PCR assay. J. Virol. Meth. 71, 46–47.
Short communication
Improving the sensitivity of single-strand conformation polymorphism (SSCP) to study the variability of PLMVd Wen Xing Xu a , Ni Hong a,c , Jian Kun Zhang a , Guo Ping Wang a,b,∗ a
College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China b Key Lab of Plant Pathology of Hubei Province, Wuhan, Hubei 430070, PR China c National Indoor Conservation Center of Virus-free Germplasms of Fruit Crops, Wuhan, Hubei 430070, PR China Received 4 December 2005; received in revised form 9 March 2006; accepted 16 March 2006 Available online 27 April 2006
Abstract Single-strand conformation polymorphism (SSCP) was used to characterize viroids. Eight cDNA clones, which showed identical profiles in preliminary existing SSCP analysis but had different sequences, were chosen to develop a sensitive SSCP technique for identifying the variability of Peach latent mosaic viroid (PLMVd). Polyacrylamide Gel Electrophoresis (PAGE) conditions were optimized to improve the sensitivity of the existing SSCP, and a modified SSCP protocol was developed. The results indicated that the modified SSCP protocol provided an overall sensitivity in identifying the variability of these clones, and showed higher resolution than the existing one and its improved versions. As shown by sequence analyses of cDNA clones of PLMVd and the modified SSCP profiles, there is no close correlation between the number of base changes and variation of the modified SSCP band patterns. The potential use of the modified SSCP analysis is discussed as a tool for viroids characterization. © 2006 Elsevier B.V. All rights reserved. Keywords: Peach latent mosaic viroid; SSCP
Viroids, the smallest infectious agents in plants, have singlestrand circular RNA genomes (245–401 nucleotides), and do not have a mRNA capacity and must recruit host proteins to assist in the steps of life cycle (Symons, 1997; Pelchat et al., 2003). Sequence variability has been reported in different viroid populations, and shows the high plasticity of these minimal genetic systems (Koltunow and Rezaian, 1988; Diener, 1996; Kofalvi et al., 1997). Many studies indicated that the change of a few nucleotides or even one nucleotide in viroid genomes could change dramatically their virulence to the host (Dickson et al., 1979; Visvader and Symons, 1985; Hammond, 1992; Reanwarakorn and Semancik, 1998; Qi and Ding, 2003). Therefore, it is necessary to develop a highly sensitive technique for understanding the relationship of molecular variation among viroids and their virulence. The single-strand conformation polymorphism (SSCP) technique is a feasible method for detection of point mutations and DNA polymorphisms (Orita et al., 1989a,b). Because of
∗
Corresponding author. Tel.: +86 27 87282590; fax: +86 27 87397735. E-mail address: [email protected] (G.P. Wang).
0166-0934/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2006.03.004
its speed, simplicity and applicability to large-scale screening, SSCP has been applied in plant virology to study molecular variability and molecular epidemiology, and to detect mixed infections (Rubio et al., 1996; Magome et al., 1999; Kong et al., 2000). SSCP has also been used to characterize Potato spindle tuber viroid, Citrus exocortis viroid and some other viroids, and the results have demonstrated that SSCP may be suitable for routine analysis of variability of their unknown sequences when the gel and electrophoresis conditions were optimized (Chen et al., 1995; Szychowski et al., 1998; Palacio and Duran-Vila, 1999; Foissac and Duran-Vila, 2000; Palacio-Bielsa et al., 2003). In this study, SSCP was used to study the genetic diversity of Peach latent mosaic viroid (PLMVd), a typical species of the genus Pelamoviroid, but the existing SSCP is not sensitive enough to distinguish small differences among different variants, even after changing several important parameters of the technique. A modified method is discussed based on the existing SSCP to study the variability of PLMVd. Young leaves of peach infected with viroids were collected from eight peach plants of the cultivar ‘Yuhualu’ with symptom of discoloration of peach latent mosaic disease in an orchard in spring, in the Hubei Province of China.
W.X. Xu et al. / Journal of Virological Methods 135 (2006) 276–280
Viroid cRNA was extracted according to the method of Zhang et al. (1998). The extracted viroid RNA was subjected directly to reverse transcription. The first-strand cDNA was synthesized using Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (Promega, Madison, WI) with primer1 (5 -ATCACACCCTCCTCGGAACCAA-3 ) comple-
277
mentary to the RNA sequence of PLMVd-P3 at 181–202 nt (Xu et al., 2005). Double-strand DNA was amplified with primer1 and primer2 (5 -CCAGGTAACGCCGTAGAAACTG3 ), which was homologous to the RNA sequence of PLMVd-P3 at 203–224 nt. PCR amplification was carried out using a thermal cycler (PTC-200; MJ Research Inc, USA) for 35 cycles of
Fig. 1. Alignment of sequences representing clone V1–V8.
278
W.X. Xu et al. / Journal of Virological Methods 135 (2006) 276–280
94 ◦ C/45 s and 60 ◦ C/45 s and 72 ◦ C/45 s after denaturation for 3 min in 94 ◦ C, and followed by a final extension of 7 min at 72 ◦ C. PCR products were subjected to electrophoresis in 1% agarose gel to confirm the synthesis of a DNA product of the expected size. The full-size PLMVd DNA of each sample was gel purified using agarose gel DNA purification kit (Takara Corporation, Dalian, PR China) and ligated to vector pMD18-T, followed by transformation into Escherichia coli DH5␣. The existing SSCP was carried out according to the method of Palacio and Duran-Vila (1999). Briefly, aliquots of 5 l of the RT-PCR products amplified from nucleic acid preparations were mixed with 8 l denaturing loading buffer (95% formamide, 10 mM NaOH, 0.05% bromphenol blue, 0.05% xylene cyanol), or aliquots of 0.2 l of the PCR products amplified from purified plasmids mixed with 2 l denaturing loading buffer, heated for 8 min at 100 ◦ C and chilled on ice for 3 min, then loaded into pre-run (200 V for 10 min) and pre-cooled (in 4 ◦ C for 30 min) 14% non-denaturing polyacrylamide (acrylamide:bisacrylamide, 49:1) gel (20 cm × 12.5 cm × 0.1 cm). Electrophoresis was carried out at 200 V in a refrigerator at 4 ◦ C in 1× TBE buffer (89 mM Tris, 89 mM boric acid, 2.5 mM EDTA, pH 8.3) for 2 h. The gel was then stained with silver according to the method of Igloi (1983). Using the existing SSCP system, eight samples of RT-PCR products amplified from nucleic acid preparations had eight identical profiles, as did eight clones (V1–V8) selected at random from these samples (data not shown). To understand the resolution of the existing SSCP, the eight recombinant plasmids were purified and sequenced with ABI PRISM DNA sequencer 377 (Perkin-Elmer). The sequences were analyzed using the Clustal V program (Thomson et al., 1994). The sequencing results indicated the eight clones showed changes from 1 to 25 nucleotides, including transitions, transversions, additions and deletions and covered most variable sites in the viroid and their homologies ranged from 99.7% to 92.4% (Fig. 1), which means the resolution of existing SSCP was very low. Some PAGE conditions were assayed to improve the resolution of the existing SSCP using the eight clones: (a) electrophoresis time (2 and 16 h); (b) gel concentration (14% and 8%); (c) running voltage (200 and 300 V); (d) cross-linking ratio of acrylamide and bisacrylamide (49:1 and 29:1). Based on these conditions, a modified SSCP was designed and the gel was prepared with two equal sections: the upper section was 8% polyacrylamide with a cross-linking ratio of 49:1, the bottom section was 8% polyacrylamide with a cross-linking ratio of 29:1. About 100 l isopropylalcohol was used to obtain a good horizontal cross-linked line of two parts of gel, and to cover the surface of the bottom part of gel before solidification and then removed after gel solidification, and the upper section gel was then prepared. Electrophoresis was carried out at 200 V for 16 h. Analysis of the profiles of electrophoresis indicated that prolonging the electrophoresis time from 2 to 16 h in 14% PAGE gel still had a low resolution. Among the eight clones analyzed, only clone V6–V8 showed different profiles with each other, but they could not be distinguished easily when they were mixed together, one clone remained undetected (Figs. 2A, 3A, 4A). When the gel concentration was reduced to 8% and other param-
Fig. 2. SSCP profiles of clone V1–V3 and mixed sample of the three clones in 14% PAGE gel with a cross-linking ratio of 49:1 at 200 V for 16 h (A). The same samples analyzed in 8% PAGE gel with a cross-linking ratio of 49:1 (B) and a cross-linking ratio of 29:1 (C) at 200 V for 16 h, and in 8% PAGE gel with a cross-linking ratio of 49:1 at 300 V (D), and in modified SSCP (E).
eters were unchanged, clone V1 could be discriminated with V2 and V3, but clone V2 and V3 showed same band migrations and still could not be discriminated from each other, and one clone remained undetected from mixed sample of V1–V3 (Fig. 2B). Mixed sample of V4 and V5 showed a three-band pattern, the upper two bands of them could not be differentiated (Fig. 3B). Under the same gel concentration, the changed voltage (300 V) and gel cross-linking ratio (29:1) could not give a higher resolution for mixed sample of V1–V3 (Fig. 2C and D), so did changed gel cross-linking ratio for mixed sample of V4 and V5 (Fig. 3C). In the modified SSCP analysis, the resolution was greatly improved, V1–V3 in single or mixed sample was clearly discriminated on the gel (Fig. 2E), so did V4 and V5 (Fig. 3D), V6–V8 (Fig. 4B). All eight clones examined showed different profiles and all the variations in these clones were dis-
W.X. Xu et al. / Journal of Virological Methods 135 (2006) 276–280
279
Fig. 4. SSCP profiles of clone V6–V8 and mixed sample of the three clones in 14% PAGE gel with a cross-linking ratio of 49:1 at 200 V for 16 h (A) and in modified SSCP (B).
Fig. 3. SSCP profiles of clone V4, V5 and mixed sample of the two clones in 14% PAGE gel with a cross-linking ratio of 49:1 at 200 V for 16 h (A). The same samples analyzed in 8% PAGE gel with a cross-linking ratio of 49:1 (B) and a cross-linking ratio of 29:1 (C), and in modified SSCP (D).
tinguished by the modified SSCP. Even a single base variation (T↔C in nucleotide 27) in variants V4 and V5 was clearly identified (Fig. 3D). No correlation existed between the number of base changes and variation of the SSCP band patterns, as reported previously by Palacio and Duran-Vila (1999). For example, total 12 base exchanges in V7 and V8 gave them more obviously distinct band migration than 17 base changes giving V2 and V3 in modified SSCP (lanes 2 and 3 of Figs. 2E and 4B).
The modified SSCP gave an overall sensitivity in identifying the variability of the eight clones and showed a higher resolution capability than the existing one and its improved versions. The modified SSCP with two cross-linking ratio of acrylamide and bisacrylamide within a gel summarized the resolution capacity of two conditions and gave an increased sensitivity. Clones such as V1 and V2 could not be distinguished in 8% gel with a crosslinking ratio of 29:1, but they could be distinguished in 8% gel with a cross-linking ratio of 49:1; clone V2 and V3 were on the contrary (Fig. 2B and C), when such two conditions were combined together, these samples could be distinguished in one test (Fig. 2E). Whether the modified SSCP could provide sufficient sensitivity to identify all existing sequence heterogeneity in complex mixtures of DNA samples needs further study. As a more sensitive variability identifying protocol the existing one, the modified SSCP analysis is not only used as a complementary tool in viroid characterization studies in which extensive sequencing is required, it could also provide valuable information for identification of molecular variability of viroid or RNA genomes, as well as for screening molecular variants, detection of mixed infections, comparison of field isolates. Acknowledgements This study was supported by National 863 Project of China (No. 2001AA241142). The authors would like to thank Profes-
280
W.X. Xu et al. / Journal of Virological Methods 135 (2006) 276–280
sor DaoHong Jiang, College of Plant Science and Technology, Huazhong Agricultural University, and Professor ShiYun Chen, Wuhan Institute of Virology, the Chinese Academy of Sciences for reading the manuscript and giving valuable suggestions. References Chen, X., Baumstark, T., Steger, G., Riesner, D., 1995. High resolution SSCP by optimization in the temperature by transverse TGGE. Nucleic Acids Res. 23, 4524–4525. Dickson, E., Robertson, H.D., Niblett, C.L., Horst, R.K., Zaitlin, M., 1979. Minor differences between nucleotide sequences of mild and severe strains of potato spindle tuber viroid. Nature 277, 60–62. Diener, T.O., 1996. Origin and evolution of viroids and viroid-like satellite RNAs. Virus Genes 11, 47–59. Foissac, X., Duran-Vila, N., 2000. Characterisation of two citrus apscaviroids isolated in Spain. Arch. Virol. 145, 1975–1983. Hammond, R.W., 1992. Analysis of the virulence modulating region of potato spindle tuber viroid (PSTVd) by site mutagenesis. Virology 187, 654– 662. Igloi, G.L., 1983. Silver stain for detection of nanogram amounts of tRNA following two-dimensional electrophoresis. Anal. Biochem. 134, 184–188. Kofalvi, S.A., Marcos, J.F., Ca˜nizares, M.C., Pall´as, V., Candresse, T., 1997. Hop stunt viroid (HSVd) sequence variants from Prunus species: evidence for recombination between HSVd isolates. J. Gen. Virol. 78, 3177–3186. Koltunow, A.M., Rezaian, M.A., 1988. Grapevine yellow speckle viroid: structural features of a new viroid group. Nucleic Acids Res. 16, 849–864. Kong, P., Rubio, L., Polek, M., Falk, B.W., 2000. Population structure and genetic diversity within California citrus tristeza virus (CTV) isolates. Virus Genes 21, 139–145. Magome, H., Yoshikawa, N., Takahashi, T., 1999. Single-strand conformation polymorphism analysis of apple stem grooving capillovirus sequence variants. Phytopathology 89, 136–140. Orita, M., Iwahana, H., Kanazawa, H., Hayashi, K., Sekiya, T., 1989a. Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc. Natl. Acad. Sci. U.S.A. 86, 2766–2770.
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