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Display of sequence variation in PCR-amplified mitochondrial DNA regions of Echinococcus by single-strand conformation polymorphism
Acta Tropica 71 (1998) 107 – 115
Display of sequence variation in PCR-amplified mitochondrial DNA regions of Echinococcus by single-strand conformation polymorphism Robin B. Gasser a,*, Xingquan Zhu a, Donald P. McManus b a
Department of Veterinary Science, The Uni6ersity of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia b Molecular Parasitology Unit, Queensland Institute of Medical Research, 300 Herston Road, Brisbane 4006, Australia Received 14 November 1997; received in revised form 9 June 1998; accepted 11 June 1998
Abstract Echinococcosis, a disease caused by infection with the larval stage of a tapeworm parasite of the genus Echinococcus, is of major socio-economic importance, and studying genetic variability within and between Echinococcus populations has important implications for disease control and epidemiology. Various DNA approaches have been used to study Echinococcus genetics, but most methods do not allow the accurate display or definition of mutational/allelic variation. To overcome this limitation, we established a mutation scanning approach. Single-strand conformation polymorphism (SSCP) of two different enzymatically amplified mitochondrial (mt) DNA regions was evaluated using seven different genotypes of Echinococcus (defined as G1, G4, G6, G8, O, V and M2). The NADH dehydrogenase 1 gene (ND1 ) or the cytochrome c oxidase subunit 1 (CO1 ) were amplified by polymerase chain reaction from parasite DNA, denatured and directly subjected to electrophoresis in a non-denaturing gel matrix. Each of the seven genotypes examined could be delineated from one another based on characteristic and reproducible banding patterns. The results demonstrate the usefulness of SSCP for the direct visual display of sequence variation in mtDNA of Echinococcus without the need for DNA sequencing or restriction analyses, and indicate
* Corresponding author. Fax: + 61 3 7410401; e-mail: [email protected] 0001-706X/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII S0001-706X(98)00052-7
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its potential for studying allelic variability in a range of other genes. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Cytochrome c oxidase subunit 1; Echinococcus; Mitochondrial DNA; NADH dehydrogenase 1; Polymerase chain reaction-based single-strand conformation polymorphism; Sequence variation
1. Introduction Human echinococcosis is a disease caused by infection with the larval stage of a tapeworm parasite belonging to the genus Echinococcus (Cestoda: Taeniidae). Studying genetic variation within and between Echinococcus populations can have significant implications for epidemiology and disease control. A range of DNA approaches has been used to study genetic variation within Echinococcus (Thompson et al., 1995, McManus and Bowles, 1996). Traditionally, restriction fragment length polymorphism (RFLP) analysis of genomic DNA by Southern blotting was used (McManus and Rishi, 1989, Hope et al., 1991). More recently, polymerase chain reaction (PCR)-based approaches (Mullis et al., 1986) such as PCR-linked RFLP and arbitrarily primed PCR (AP-PCR) (Welsh and McClelland, 1990, Williams et al., 1990) have found broad applicability because their sensitivity permits the analysis of genes from minute amounts of parasite material (e.g. Bowles et al., 1992, Bowles and McManus, 1993a,b, Siles-Lucas et al., 1993, Bowles et al., 1994, Gasser and Chilton, 1995). Unfortunately, the electrophoretic procedures (usually agarose gel electrophoresis) linked with these methods do not usually allow the accurate display or definition of mutational/allelic sequence variation, because they rely solely on the separation of DNA fragments by size. Direct nucleotide sequencing of PCR products combined with denaturing gel electrophoresis has the highest capacity to resolve sequence variability (e.g. Gasser et al., 1993), but it is laborious and costly to perform when large numbers of samples require analysis. Mutation scanning methods provide alternatives for the high resolution analysis of sequence variation in PCR products, without the need for sequencing (Cotton, 1997). Although widely used in the biomedical field, their application to parasite genes is limited (see Gasser, 1997). Single-strand conformation polymorphism (SSCP) is a simple mutation scanning method, which has the potential to discriminate DNA strands differing by a single nucleotide (Orita et al., 1989). Unlike conventional approaches, SSCP is based on the principle that the electrophoretic mobility of a single-stranded DNA molecule in a non-denaturing gel is dependent on its size and structure (i.e. conformation). The length of a strand, location and number of regions of base pairing determine the secondary and tertiary conformations of the molecule, which are highly dependent on the primary sequence. Hence, a mutation at a particular site in the primary sequence can modify the conformation of the molecule, which alters its electrophoretic mobility. In this study, we employed SSCP for the direct visual display of sequence variation in PCR-amplified
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fragments of the mitochondrial genome within the genus Echinococcus, and discuss the implications of this scanning method to study molecular variation in parasites.
2. Materials and methods
2.1. Parasite material and isolation of genomic DNA Larval or strobilate stages of Echinococcus (15 isolates) were collected from intermediate or definitive hosts from different geographical locations (Table 1). Parasites were washed extensively in physiological saline and frozen at −20°C until required. Genomic DNA was then isolated from 50–100 ml of pelleted parasite material by standard sodium dodecyl sulphate–proteinase K treatment and purified with Prep-A-Gene™ (Bio-Rad) (Gasser et al., 1993).
2.2. Enzymatic amplification Mitochondrial DNA regions, known specifically to provide markers for the different genotypes within Echinococcus, were amplified by PCR from individual samples of Echinococcus DNA (10–20 ng template) using 33P-endlabelled oligonucleotide primers. A fragment from the NADH dehydrogenase 1 gene (ND1 ) was amplified with primers JB11 (5%-AGATTCGTAAGGGGCCTAATA-3%) and JB12 (5%-ACCACTAACTAATTCACTTTC-3%) (Bowles and McManus, 1993b), and another from the cytochrome c oxidase subunit 1 (CO1 ) was amplified with primers JB3 (5%-TTTTTTGGGCATCCTGAGGTTTAT-3%) and JB4.5 (5%-TAAAGAAATable 1 DNA samples representing seven genotypes of Echinococcus Sample
Genotype a
Parasite stage, host
Geographical origin
Eg1 Eg2 Eg3 Eg4 Eg5 Eg6 Eg11 Eg12 EgH EgHpsc EgC Egcerv Eo Ev EmR
G1 G1 G1 G1 G1 G1 G1 G1 G4 G4 G6 G8 O V M2
Protoscolex, sheep Protoscolex, sheep Adult worm, dog (from Yak) Protoscolex, sheep Protoscolex, macropod Protoscolex, sheep Protoscolex, goat Protoscolex, macropod Protoscolex, horse Protoscolex, horse Protoscolex, camel Protoscolex, moose Protoscolex, rodent Protoscolex, rodent Cyst/protoscolex, vole
Douglas Station, Falkland Islands Port Louis, Falkland Islands Gansu, China Altai, China New South Wales, Australia Wales, UK Turkana, Kenya New South Wales, Australia Ireland Cheshire, UK Somalia Minnesota, USA Panama South America Hohenheim, Germany
a
Based on Bowles et al. (1992) and Bowles and McManus (1993b).
R.B. Gasser et al. / Acta Tropica 71 (1998) 107–115
Fig. 1.
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111
GAACATAATGAAAATG-3%) (Bowles et al., 1992). PCR reactions (25 ml) were performed in 10 mM Tris – HCl, pH 8.4, 50 mM KCl, 3.0 mM MgCl2; 250 mM of each dNTP, 25 pmol of each primer and 2 U Taq polymerase (AmpliTaq, Perkin Elmer) under the following conditions: 94°C for 30 s (denaturation), 55°C for 30 s (annealing), 72°C for 30 s (extension) for 35 cycles, followed by a final extension at 72°C for 5 min. For each set of PCR reactions, negative (no-DNA) and positive controls were included. Prior to SSCP analysis, individual PCR products (5 ml) were checked on ethidium bromide-stained 2.5% agarose–TBE (65 mM Tris–HCl, 22.5 mM boric acid, 1.25 mM EDTA, pH 9) gels, using pGEM™ as the size marker (Promega). Agarose gels were photographed, dried on to blotting paper and subsequently exposed to X-ray film (RP1, Agfa).
2.3. Single-strand conformation polymorphism (SSCP) A method similar to that originally described by Orita et al. (1989) was used. Ten microliters of each PCR product were mixed with an equal volume of denaturing buffer (10 mM NaOH, 95% formamide, 0.05% bromophenol blue and 0.05% xylene cyanole). After denaturation at 94°C for 5 min and subsequent snap-cooling on a freeze block (−20°C), 3 ml of individual samples were loaded into the wells of a 0.4 mm thick, 0.6× mutation detection enhancement gel (MDE, commercially available through FMC BioProducts, Rockland, MN) and subjected to electrophoresis (7 W for 15 h at 20°C) in a conventional sequencing rig (Base-Runner; IBI, New Haven, CT) using TBE as the buffer. Gels were subsequently dried on to blotting paper and subjected to autoradiography. After autoradiography, the position of a band on an SSCP gel was determined by measuring its distance (in mm) from a particular reference marker (1605; see Fig. 1) and multiplying the value by 100.
3. Results and discussion The amplification products of ND1 and CO1 were 525 bp and 460 bp in size, respectively (Fig. 1). Autoradiograhic exposure of agarose gels demonstrated the specificity of the amplification and the products (not shown). A number of vertebrate DNA samples (sheep, ox and kangaroo) was also subjected to the same Fig. 1. Single-strand conformation polymorphism (SSCP) analysis of ND1 or CO1 PCR products of Echinococcus. Panels A (ND1 ) and B (CO1 ) show SSCP patterns for genotype G1 (samples Eg1, Eg2, Eg3, Eg4, Eg5, Eg6, Eg11 and Eg12, lanes 1 – 8, respectively) using genotypes G6 (lane 9) and G4 (samples EgH and EgHpsc, lanes 10 and 11) as controls. Panels C (ND1 ) and D (CO1 ) display sequence variability among seven genotypes O, V, M2, G1 (sample Eg1), G4 (sample EgH), G6 and G8 (lanes 1 – 7, respectively). S and N represent sheep-DNA and no-DNA control lanes, respectively. On agarose gels, ‘P’ represents the pGEM™ size marker (Promega), whereas on SSCP gels it represents solely a reference (not size) marker. For individual genotypes of Echinococcus, ‘A’ indicates the positions of the ssND1 -A or ssCO1 -A bands; ‘B’ indicates the positions of the ssND1 -B or ssCO1 -B bands (see Table 2). Below each SSCP panel is an agarose gel (samples in the same order) showing the quality of the PCR products used for SSCP analysis.
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amplification procedure used for Echinococcus genomic DNA, and in no case were amplification products detected in the control samples on the autoradiographs of agarose gels (sheep DNA control shown as a representative in Fig. 1). For both ND1 and CO1, there was no detectable size variation on agarose gels among PCR products derived from the 15 samples of Echinococcus, yet previous studies have shown that there are significant sequence differences among the different genotypes within the genus (Bowles et al., 1992, Bowles and McManus, 1993b). These sequence differences in the ND1 and CO1 were exploited in SSCP for the identification of each of the seven genotypes (O, V, M2, G1, G4, G6 and G8 ) of Echinococcus by their single-strand (ss) banding patterns (Fig. 1). For both ND1 and CO1, at least two major (strong) ss bands were resolved per genotype. The resolution of 1 – 5 additional ss bands (of variable intensities) in SSCP indicated the existence of different sequence types within the amplification products and/or the formation of different conformers of ss molecule(s) (Fig. 1). For both ND1 and CO1, the ss-banding patterns among eight samples representing genotype G1 (Fig. 1A, B) were very similar, while distinct differences in the patterns were detected among the seven genotypes of Echinococcus examined (Fig. 1C, D). Individual genotypes could be delineated from one another by the relative positions of particular ssND1 and/or ssCO1 bands on SSCP gels (Table 2). For instance, five (V, M2, G1, G6 and G8 ) of the seven genotypes could be distinguished from one another and from the other two genotypes by the positions of their ssND1 -B bands (4850, 4800, 5000, 4900 and 5050, respectively); genotypes O and G4 could each be identified by the positions of their ssND1 -A bands (7200 and 7250, respectively) with reference to their ssND1 -B bands (both with position 4950) (Table 2). Using CO1, four genotypes (O, V, G6 and G8 ) could be distinguished from one another and from the other three genotypes by the positions of the ssCO1 -B bands (3500, 3450, 3250 and 3350, respectively); genotypes M2, G1 and G4 could each be identified by the positions of the ssCO1 -A bands (5650, 5750 and 5600, respectively) with reference to their ssCO1 -B bands (all with position 3350) (Table 2). For both mtDNA fragments employed, individual SSCP patterns were reproducible on different days using PCR products amplified on different days (not shown). Table 2 Relative positions of some bands on SSCP gels determined in relation to the reference marker 1605 (see Section 2 and Fig. 1) Band
ssND1 -A ssND1 -B ssCO1 -A ssCO1 -B
Genotype O
V
M2
G1
G4
G6
G8
7200 4950 5500 3500
7250 4850 5650 3450
7150 4800 5650 3350
7200 5000 5750 3350
7250 4950 5600 3350
7300 4900 5700 3250
7350 5050 5700 3350
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The ability to identify each of the seven genotypes (O, V, M2, G1, G4, G6 and G8 ) of Echinococcus by SSCP analysis of ND1 and CO1 fragments was in accordance with previous sequencing results (Bowles and McManus, 1993b). The variation in SSCP patterns among these genotypes reflected sequence differences ranging from 12.5 to 16% for ND1 and from 6 to 11.5% for CO1, figures based on partial sequence data (Bowles and McManus, 1993b). Although the PCR products of ND1 were 65 bp larger than CO1, slightly better differentiation among the genotypes of Echinococcus was achieved with ND1 in SSCP than with CO1 ; this appeared to be due to a higher level of sequence variation among the genotypes. The capacity of SSCP to delineate sequences differing by 6–16% suggests that all of the genotypes of Echinococcus thus far examined (see Bowles and McManus, 1993b) could be delineated by this mutation scanning approach. It may be possible, for example, to delineate among genotypes G1, G2 and G3 of Echinococcus granulosus, which differ in sequence by less than 1% in both subunits of ND1 and CO1 (Bowles and McManus, 1993b), because our recent work has demonstrated that SSCP can distinguish PCR-amplified ribosomal DNA fragments (530 bp) which differ by a single nucleotide (Zhu et al., 1998). Nevertheless, this requires critical testing for genotypes G1 –G3. Other studies indicate that 75– 100% of point mutations can be detected by SSCP over sequence lengths of 100 – 200 bp, but the mutation detection rate may decrease for sequences longer than 200 bp (see Cotton, 1997). If it were not possible to delineate among genotypes G1 – G3 using 460 – 525 bp mtDNA fragments under the current SSCP conditions, shorter fragments should be used and electrophoresis conditions modified. The present study demonstrates that PCR-linked SSCP provides a reliable method to display variation in mitochondrial sequences of Echinococcus, which has significant implications for studying the population genetics of this parasite. This mutation scanning strategy has several advantages over other approaches to screen for molecular variation (see Gasser, 1997). First, given that the conformations of single-stranded DNA molecules are sensitive to nucleotide changes in a sequence, SSCP can be used to screen large numbers of Echinococcus samples for allelic sequence variation without the need for PCR-RFLP or DNA sequence analysis, which reduces time, labour and expense. Also, the resolution of sequence variation in SSCP gels is superior compared with say PCR-RFLP analysis on agarose gels. In contrast to PCR-RFLP, which screens for variation in a sequence at a limited number of endonuclease restriction sites, the SSCP approach has the capacity to scan an entire sequence for variation, albeit over a relatively short length. Moreover, in contrast to AP-PCR methods (see Ellsworth et al., 1993, MacPherson et al., 1993), this approach employs specific primers at high stringency in PCR, thus minimising the possibility of co-amplification of contaminating DNA and maximising reproducibility. Although SSCP has been evaluated in this study for the direct display of variation in mtDNA sequences of Echinococcus, it has the potential to be used to study sequence variation in any gene of any parasite. This has important implications for addressing fundamental aspects relating to the taxonomy of parasites, the genetic structure of parasite populations, and the organisation of parasite genes.
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Acknowledgements The authors thank M.P. Reichel (New Zealand), P.S. Craig (UK), R.M. Martin and D.J. Jenkins (Australia) for providing some of the isolates used in this study. Project funding was provided in part by the Australia Research Council Collaborative Research Program (The University of Melbourne) (R.G.), the Department of Industry, Science and Tourism and the National Health and Medical Research Council (D.M.). X.Z. is a recipient of scholarships through the University of Melbourne.
References Bowles, J., McManus, D.P., 1993a. Rapid discrimination of Echinococcus species and strains using a polymerase chain reaction-based RFLP method. Mol. Biochem. Parasitol. 57, 231 – 240. Bowles, J., McManus, D.P., 1993b. NADH dehydrogenase 1 gene sequences compared for species and strains of the genus Echinococcus. Int. J. Parasitol. 23, 969 – 972. Bowles, J., Blair, D., McManus, D.P., 1992. Genetic variants within the genus Echinococcus identified by mitochondrial DNA sequencing. Mol. Biochem. Parasitol. 54, 165 – 174. Bowles, J., Blair, D., McManus, D.P., 1994. Molecular genetic characterization of the cervid strain (‘northern form’) of Echinococcus granulosus. Parasitology 109, 215 – 221. Cotton, R.G.H., 1997. Mutation Detection. Oxford University Press, Oxford. Ellsworth, D.L., Rittenhouse, K.D., Honeycutt, R.L., 1993. Artifactual variation in randomly amplified polymorphic DNA banding patterns. BioTechniques 14, 214 – 217. Gasser, R.B., 1997. Mutation scanning methods for the analysis of parasite genes. Int. J. Parasitol. 27, 1449–1463. Gasser, R.B., Chilton, N.B., 1995. Characterisation of taeniid cestode species by PCR-RFLP of ITS2 ribosomal DNA. Acta Trop. 59, 31–40. Gasser, R.B., Chilton, N.B., Hoste, H., Beveridge, I., 1993. Rapid sequencing of rDNA from single worms and eggs of parasitic helminths. Nucleic Acids Res. 21, 2525 – 2526. Hope, M., Bowles, J., McManus, D.P., 1991. A reconsideration of the Echinococcus granulosus strain situation in Australia following RFLP analysis of cystic material. Int. J. Parasitol. 21, 471 – 475. MacPherson, J.M., Eckstein, P.E., Scoles, G.J., Gajadar, A.A., 1993. Variability of the random amplified polymorphic DNA assay among thermal cyclers, and effects of primer and DNA concentration. Mol. Cell. Probes 7, 293 – 299. McManus, D.P., Bowles, J., 1996. Molecular genetic approaches to parasite identification: their value in diagnostic parasitology and systematics. Int. J. Parasitol. 26, 687 – 704. McManus, D.P., Rishi, A.K., 1989. Genetic heterogeneity within Echinococcus granulosus: isolates from different hosts and geographical areas characterized with DNA probes. Parasitology 99, 17 – 29. Mullis, K.B., Faloona, F., Scharf, S., Saiki, R., Horn, G., Erlich, H., 1986. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symp. Quant. Biol. 51, 263–273. Orita, M., Suzuki, Y., Sekiya, T., Hayashi, K., 1989. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5, 874 – 879. Siles-Lucas, M., Cuesta-Bandera, C., Cesar-Benito, M., 1993. Random amplified polymorphic DNA technique for speciation studies of Echinococcus granulosus. Parasitol. Res. 79, 343 – 345. Thompson, R.C.A., Lymbery, A.J., Constantine, C.C., 1995. Variation in Echinococcus: towards a taxonomic revision of the genus. Adv. Parasitol. 35, 146 – 176. Welsh, J., McClelland, M., 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18, 7213–7218.
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Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A., Tingey, S.V., 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18, 6531 – 6535. Zhu, X.Q., Jacobs, D.E., Chilton, N.B., Sani, R.A., Cheng, N.A.B.Y., Gasser, R.B., 1998. Molecular characterization of a Toxocara variant from cats in Kuala Lumpur, Malaysia. Parasitology (in press).
.
Display of sequence variation in PCR-amplified mitochondrial DNA regions of Echinococcus by single-strand conformation polymorphism Robin B. Gasser a,*, Xingquan Zhu a, Donald P. McManus b a
Department of Veterinary Science, The Uni6ersity of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia b Molecular Parasitology Unit, Queensland Institute of Medical Research, 300 Herston Road, Brisbane 4006, Australia Received 14 November 1997; received in revised form 9 June 1998; accepted 11 June 1998
Abstract Echinococcosis, a disease caused by infection with the larval stage of a tapeworm parasite of the genus Echinococcus, is of major socio-economic importance, and studying genetic variability within and between Echinococcus populations has important implications for disease control and epidemiology. Various DNA approaches have been used to study Echinococcus genetics, but most methods do not allow the accurate display or definition of mutational/allelic variation. To overcome this limitation, we established a mutation scanning approach. Single-strand conformation polymorphism (SSCP) of two different enzymatically amplified mitochondrial (mt) DNA regions was evaluated using seven different genotypes of Echinococcus (defined as G1, G4, G6, G8, O, V and M2). The NADH dehydrogenase 1 gene (ND1 ) or the cytochrome c oxidase subunit 1 (CO1 ) were amplified by polymerase chain reaction from parasite DNA, denatured and directly subjected to electrophoresis in a non-denaturing gel matrix. Each of the seven genotypes examined could be delineated from one another based on characteristic and reproducible banding patterns. The results demonstrate the usefulness of SSCP for the direct visual display of sequence variation in mtDNA of Echinococcus without the need for DNA sequencing or restriction analyses, and indicate
* Corresponding author. Fax: + 61 3 7410401; e-mail: [email protected] 0001-706X/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII S0001-706X(98)00052-7
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its potential for studying allelic variability in a range of other genes. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Cytochrome c oxidase subunit 1; Echinococcus; Mitochondrial DNA; NADH dehydrogenase 1; Polymerase chain reaction-based single-strand conformation polymorphism; Sequence variation
1. Introduction Human echinococcosis is a disease caused by infection with the larval stage of a tapeworm parasite belonging to the genus Echinococcus (Cestoda: Taeniidae). Studying genetic variation within and between Echinococcus populations can have significant implications for epidemiology and disease control. A range of DNA approaches has been used to study genetic variation within Echinococcus (Thompson et al., 1995, McManus and Bowles, 1996). Traditionally, restriction fragment length polymorphism (RFLP) analysis of genomic DNA by Southern blotting was used (McManus and Rishi, 1989, Hope et al., 1991). More recently, polymerase chain reaction (PCR)-based approaches (Mullis et al., 1986) such as PCR-linked RFLP and arbitrarily primed PCR (AP-PCR) (Welsh and McClelland, 1990, Williams et al., 1990) have found broad applicability because their sensitivity permits the analysis of genes from minute amounts of parasite material (e.g. Bowles et al., 1992, Bowles and McManus, 1993a,b, Siles-Lucas et al., 1993, Bowles et al., 1994, Gasser and Chilton, 1995). Unfortunately, the electrophoretic procedures (usually agarose gel electrophoresis) linked with these methods do not usually allow the accurate display or definition of mutational/allelic sequence variation, because they rely solely on the separation of DNA fragments by size. Direct nucleotide sequencing of PCR products combined with denaturing gel electrophoresis has the highest capacity to resolve sequence variability (e.g. Gasser et al., 1993), but it is laborious and costly to perform when large numbers of samples require analysis. Mutation scanning methods provide alternatives for the high resolution analysis of sequence variation in PCR products, without the need for sequencing (Cotton, 1997). Although widely used in the biomedical field, their application to parasite genes is limited (see Gasser, 1997). Single-strand conformation polymorphism (SSCP) is a simple mutation scanning method, which has the potential to discriminate DNA strands differing by a single nucleotide (Orita et al., 1989). Unlike conventional approaches, SSCP is based on the principle that the electrophoretic mobility of a single-stranded DNA molecule in a non-denaturing gel is dependent on its size and structure (i.e. conformation). The length of a strand, location and number of regions of base pairing determine the secondary and tertiary conformations of the molecule, which are highly dependent on the primary sequence. Hence, a mutation at a particular site in the primary sequence can modify the conformation of the molecule, which alters its electrophoretic mobility. In this study, we employed SSCP for the direct visual display of sequence variation in PCR-amplified
R.B. Gasser et al. / Acta Tropica 71 (1998) 107–115
109
fragments of the mitochondrial genome within the genus Echinococcus, and discuss the implications of this scanning method to study molecular variation in parasites.
2. Materials and methods
2.1. Parasite material and isolation of genomic DNA Larval or strobilate stages of Echinococcus (15 isolates) were collected from intermediate or definitive hosts from different geographical locations (Table 1). Parasites were washed extensively in physiological saline and frozen at −20°C until required. Genomic DNA was then isolated from 50–100 ml of pelleted parasite material by standard sodium dodecyl sulphate–proteinase K treatment and purified with Prep-A-Gene™ (Bio-Rad) (Gasser et al., 1993).
2.2. Enzymatic amplification Mitochondrial DNA regions, known specifically to provide markers for the different genotypes within Echinococcus, were amplified by PCR from individual samples of Echinococcus DNA (10–20 ng template) using 33P-endlabelled oligonucleotide primers. A fragment from the NADH dehydrogenase 1 gene (ND1 ) was amplified with primers JB11 (5%-AGATTCGTAAGGGGCCTAATA-3%) and JB12 (5%-ACCACTAACTAATTCACTTTC-3%) (Bowles and McManus, 1993b), and another from the cytochrome c oxidase subunit 1 (CO1 ) was amplified with primers JB3 (5%-TTTTTTGGGCATCCTGAGGTTTAT-3%) and JB4.5 (5%-TAAAGAAATable 1 DNA samples representing seven genotypes of Echinococcus Sample
Genotype a
Parasite stage, host
Geographical origin
Eg1 Eg2 Eg3 Eg4 Eg5 Eg6 Eg11 Eg12 EgH EgHpsc EgC Egcerv Eo Ev EmR
G1 G1 G1 G1 G1 G1 G1 G1 G4 G4 G6 G8 O V M2
Protoscolex, sheep Protoscolex, sheep Adult worm, dog (from Yak) Protoscolex, sheep Protoscolex, macropod Protoscolex, sheep Protoscolex, goat Protoscolex, macropod Protoscolex, horse Protoscolex, horse Protoscolex, camel Protoscolex, moose Protoscolex, rodent Protoscolex, rodent Cyst/protoscolex, vole
Douglas Station, Falkland Islands Port Louis, Falkland Islands Gansu, China Altai, China New South Wales, Australia Wales, UK Turkana, Kenya New South Wales, Australia Ireland Cheshire, UK Somalia Minnesota, USA Panama South America Hohenheim, Germany
a
Based on Bowles et al. (1992) and Bowles and McManus (1993b).
R.B. Gasser et al. / Acta Tropica 71 (1998) 107–115
Fig. 1.
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111
GAACATAATGAAAATG-3%) (Bowles et al., 1992). PCR reactions (25 ml) were performed in 10 mM Tris – HCl, pH 8.4, 50 mM KCl, 3.0 mM MgCl2; 250 mM of each dNTP, 25 pmol of each primer and 2 U Taq polymerase (AmpliTaq, Perkin Elmer) under the following conditions: 94°C for 30 s (denaturation), 55°C for 30 s (annealing), 72°C for 30 s (extension) for 35 cycles, followed by a final extension at 72°C for 5 min. For each set of PCR reactions, negative (no-DNA) and positive controls were included. Prior to SSCP analysis, individual PCR products (5 ml) were checked on ethidium bromide-stained 2.5% agarose–TBE (65 mM Tris–HCl, 22.5 mM boric acid, 1.25 mM EDTA, pH 9) gels, using pGEM™ as the size marker (Promega). Agarose gels were photographed, dried on to blotting paper and subsequently exposed to X-ray film (RP1, Agfa).
2.3. Single-strand conformation polymorphism (SSCP) A method similar to that originally described by Orita et al. (1989) was used. Ten microliters of each PCR product were mixed with an equal volume of denaturing buffer (10 mM NaOH, 95% formamide, 0.05% bromophenol blue and 0.05% xylene cyanole). After denaturation at 94°C for 5 min and subsequent snap-cooling on a freeze block (−20°C), 3 ml of individual samples were loaded into the wells of a 0.4 mm thick, 0.6× mutation detection enhancement gel (MDE, commercially available through FMC BioProducts, Rockland, MN) and subjected to electrophoresis (7 W for 15 h at 20°C) in a conventional sequencing rig (Base-Runner; IBI, New Haven, CT) using TBE as the buffer. Gels were subsequently dried on to blotting paper and subjected to autoradiography. After autoradiography, the position of a band on an SSCP gel was determined by measuring its distance (in mm) from a particular reference marker (1605; see Fig. 1) and multiplying the value by 100.
3. Results and discussion The amplification products of ND1 and CO1 were 525 bp and 460 bp in size, respectively (Fig. 1). Autoradiograhic exposure of agarose gels demonstrated the specificity of the amplification and the products (not shown). A number of vertebrate DNA samples (sheep, ox and kangaroo) was also subjected to the same Fig. 1. Single-strand conformation polymorphism (SSCP) analysis of ND1 or CO1 PCR products of Echinococcus. Panels A (ND1 ) and B (CO1 ) show SSCP patterns for genotype G1 (samples Eg1, Eg2, Eg3, Eg4, Eg5, Eg6, Eg11 and Eg12, lanes 1 – 8, respectively) using genotypes G6 (lane 9) and G4 (samples EgH and EgHpsc, lanes 10 and 11) as controls. Panels C (ND1 ) and D (CO1 ) display sequence variability among seven genotypes O, V, M2, G1 (sample Eg1), G4 (sample EgH), G6 and G8 (lanes 1 – 7, respectively). S and N represent sheep-DNA and no-DNA control lanes, respectively. On agarose gels, ‘P’ represents the pGEM™ size marker (Promega), whereas on SSCP gels it represents solely a reference (not size) marker. For individual genotypes of Echinococcus, ‘A’ indicates the positions of the ssND1 -A or ssCO1 -A bands; ‘B’ indicates the positions of the ssND1 -B or ssCO1 -B bands (see Table 2). Below each SSCP panel is an agarose gel (samples in the same order) showing the quality of the PCR products used for SSCP analysis.
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amplification procedure used for Echinococcus genomic DNA, and in no case were amplification products detected in the control samples on the autoradiographs of agarose gels (sheep DNA control shown as a representative in Fig. 1). For both ND1 and CO1, there was no detectable size variation on agarose gels among PCR products derived from the 15 samples of Echinococcus, yet previous studies have shown that there are significant sequence differences among the different genotypes within the genus (Bowles et al., 1992, Bowles and McManus, 1993b). These sequence differences in the ND1 and CO1 were exploited in SSCP for the identification of each of the seven genotypes (O, V, M2, G1, G4, G6 and G8 ) of Echinococcus by their single-strand (ss) banding patterns (Fig. 1). For both ND1 and CO1, at least two major (strong) ss bands were resolved per genotype. The resolution of 1 – 5 additional ss bands (of variable intensities) in SSCP indicated the existence of different sequence types within the amplification products and/or the formation of different conformers of ss molecule(s) (Fig. 1). For both ND1 and CO1, the ss-banding patterns among eight samples representing genotype G1 (Fig. 1A, B) were very similar, while distinct differences in the patterns were detected among the seven genotypes of Echinococcus examined (Fig. 1C, D). Individual genotypes could be delineated from one another by the relative positions of particular ssND1 and/or ssCO1 bands on SSCP gels (Table 2). For instance, five (V, M2, G1, G6 and G8 ) of the seven genotypes could be distinguished from one another and from the other two genotypes by the positions of their ssND1 -B bands (4850, 4800, 5000, 4900 and 5050, respectively); genotypes O and G4 could each be identified by the positions of their ssND1 -A bands (7200 and 7250, respectively) with reference to their ssND1 -B bands (both with position 4950) (Table 2). Using CO1, four genotypes (O, V, G6 and G8 ) could be distinguished from one another and from the other three genotypes by the positions of the ssCO1 -B bands (3500, 3450, 3250 and 3350, respectively); genotypes M2, G1 and G4 could each be identified by the positions of the ssCO1 -A bands (5650, 5750 and 5600, respectively) with reference to their ssCO1 -B bands (all with position 3350) (Table 2). For both mtDNA fragments employed, individual SSCP patterns were reproducible on different days using PCR products amplified on different days (not shown). Table 2 Relative positions of some bands on SSCP gels determined in relation to the reference marker 1605 (see Section 2 and Fig. 1) Band
ssND1 -A ssND1 -B ssCO1 -A ssCO1 -B
Genotype O
V
M2
G1
G4
G6
G8
7200 4950 5500 3500
7250 4850 5650 3450
7150 4800 5650 3350
7200 5000 5750 3350
7250 4950 5600 3350
7300 4900 5700 3250
7350 5050 5700 3350
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The ability to identify each of the seven genotypes (O, V, M2, G1, G4, G6 and G8 ) of Echinococcus by SSCP analysis of ND1 and CO1 fragments was in accordance with previous sequencing results (Bowles and McManus, 1993b). The variation in SSCP patterns among these genotypes reflected sequence differences ranging from 12.5 to 16% for ND1 and from 6 to 11.5% for CO1, figures based on partial sequence data (Bowles and McManus, 1993b). Although the PCR products of ND1 were 65 bp larger than CO1, slightly better differentiation among the genotypes of Echinococcus was achieved with ND1 in SSCP than with CO1 ; this appeared to be due to a higher level of sequence variation among the genotypes. The capacity of SSCP to delineate sequences differing by 6–16% suggests that all of the genotypes of Echinococcus thus far examined (see Bowles and McManus, 1993b) could be delineated by this mutation scanning approach. It may be possible, for example, to delineate among genotypes G1, G2 and G3 of Echinococcus granulosus, which differ in sequence by less than 1% in both subunits of ND1 and CO1 (Bowles and McManus, 1993b), because our recent work has demonstrated that SSCP can distinguish PCR-amplified ribosomal DNA fragments (530 bp) which differ by a single nucleotide (Zhu et al., 1998). Nevertheless, this requires critical testing for genotypes G1 –G3. Other studies indicate that 75– 100% of point mutations can be detected by SSCP over sequence lengths of 100 – 200 bp, but the mutation detection rate may decrease for sequences longer than 200 bp (see Cotton, 1997). If it were not possible to delineate among genotypes G1 – G3 using 460 – 525 bp mtDNA fragments under the current SSCP conditions, shorter fragments should be used and electrophoresis conditions modified. The present study demonstrates that PCR-linked SSCP provides a reliable method to display variation in mitochondrial sequences of Echinococcus, which has significant implications for studying the population genetics of this parasite. This mutation scanning strategy has several advantages over other approaches to screen for molecular variation (see Gasser, 1997). First, given that the conformations of single-stranded DNA molecules are sensitive to nucleotide changes in a sequence, SSCP can be used to screen large numbers of Echinococcus samples for allelic sequence variation without the need for PCR-RFLP or DNA sequence analysis, which reduces time, labour and expense. Also, the resolution of sequence variation in SSCP gels is superior compared with say PCR-RFLP analysis on agarose gels. In contrast to PCR-RFLP, which screens for variation in a sequence at a limited number of endonuclease restriction sites, the SSCP approach has the capacity to scan an entire sequence for variation, albeit over a relatively short length. Moreover, in contrast to AP-PCR methods (see Ellsworth et al., 1993, MacPherson et al., 1993), this approach employs specific primers at high stringency in PCR, thus minimising the possibility of co-amplification of contaminating DNA and maximising reproducibility. Although SSCP has been evaluated in this study for the direct display of variation in mtDNA sequences of Echinococcus, it has the potential to be used to study sequence variation in any gene of any parasite. This has important implications for addressing fundamental aspects relating to the taxonomy of parasites, the genetic structure of parasite populations, and the organisation of parasite genes.
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Acknowledgements The authors thank M.P. Reichel (New Zealand), P.S. Craig (UK), R.M. Martin and D.J. Jenkins (Australia) for providing some of the isolates used in this study. Project funding was provided in part by the Australia Research Council Collaborative Research Program (The University of Melbourne) (R.G.), the Department of Industry, Science and Tourism and the National Health and Medical Research Council (D.M.). X.Z. is a recipient of scholarships through the University of Melbourne.
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