- Email: [email protected]
A simple method for cloning the complete begomovirus genome using the bacteriophage φ29 DNA polymerase
Journal of Virological Methods 116 (2004) 209–211
Short communication
A simple method for cloning the complete begomovirus genome using the bacteriophage 29 DNA polymerase Alice K. Inoue-Nagata a,∗ , Leonardo C. Albuquerque a,b , Wesley B. Rocha a,b , Tatsuya Nagata b a
b
Embrapa Hortaliças, Virology, Km. 9, BR060, C. Postal 218, 70359-970 Bras´ılia, DF, Brazil Ciˆencias Biológicas, Universidade Católica de Bras´ılia, W5 Norte, 70790-160 Bras´ılia, DF, Brazil
Received 29 June 2003; received in revised form 13 November 2003; accepted 27 November 2003
Abstract The bacteriophage DNA polymerase amplifies circular DNA in a rolling circle amplification mechanism. This characteristic was applied to amplify and clone the complete circular DNA genome of a begomovirus. Total DNA extracted from infected tissue was used as the template of an amplification reaction using the commercial kit TempliPhi (Amersham Biosciences). The amplified DNA could be used for direct sequencing and was cloned after digestion with a single cutting restriction endonuclease. The use of this enzyme simplified the cloning steps and increased the cloning efficiency of the complete genome of a circular plant DNA virus. © 2003 Elsevier B.V. All rights reserved. Keywords: Circular DNA; Geminiviridae; Rolling circle
Begomoviruses (family Geminiviridae, genus Begomovirus) are circular single-stranded DNA viruses. Members of this genus include monopartite or bipartite viruses (Van Regenmortel et al., 2000). Two methods are used commonly to clone the genome of these viruses. The first method is based on PCR using universal or specific primers, by amplifying either partially or completely the genome (Patel et al., 1993). The second method is based on the extraction of total DNA or semi-purified DNA enriched with the replicative form of the viral genome, followed by digestion with restriction enzymes, Southern blot hybridization and cloning after digestion with a single cutter enzyme (Gilbertson et al., 1991; Srivastava et al., 1995). This procedure usually has low efficiency due to the low concentration of viral genome in the preparation. A simple method is described based on the amplification of the circular DNA using a new commercial kit, TempliPhi (Amersham Biosciences), by a rolling circle amplification using the bacteriophage 29 DNA polymerase. TempliPhi
∗
Corresponding author. Tel.: +55-61-3859067; fax: +55-61-5565744. E-mail address: [email protected] (A.K. Inoue-Nagata).
0166-0934/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2003.11.015
uses an isothermal method for the exponential amplification of circular DNA. The template used for this method was a bipartite begomovirus, DF-BR2, that was found infecting tomato plants in Brazil (Inoue-Nagata et al., 2001). The DNA genome A component (DNA-A) of this virus was already cloned using the traditional method of replicative form isolation. However, the DNA genome B component (DNA-B) could not be successfully cloned, possibly due to the low concentration of this component in the infected tissue. Total DNA was extracted from infected tomato plants (Dellaporta and Hicks, 1983) and subjected to TempliPhi amplification essentially according to the manufacturer’s instructions (Amersham Biosciences). In brief, 5 l of sample buffer and 5 l of reaction buffer was added to 0.5 l of extracted DNA (ca. 0.7 g); to this mixture 0.2 l of enzyme mix was added and incubated at 30 ◦ C for approximately 20 h. Denaturation of the DNA was not essential, since the yield of the amplified DNA fragment did not vary when this step was eliminated (data not shown). The amplified DNA formed a single high molecular weight band in an agarose gel (lane 1, l l of the reaction in Fig. 1A). The DNA (1 l) was digested independently with selected restriction endonucleases and separated in
210
A.K. Inoue-Nagata et al. / Journal of Virological Methods 116 (2004) 209–211
Fig. 1. Agarose gel electrophoresis (0.7%) of amplified DNA digested with restriction enzymes (A), Southern blotting with DNA-B (B) and DNA-A (C) specific probes; 1 kb ladder (Invitrogen) was used as a marker (M). Digestion was done with the following enzymes: non-digested (1), EcoR V (2), Spe I (3), BamH I (4), EcoR I (5) and Xho I (6). Arrowhead in (A) shows unit length DNA; in (B) and (C): ND (non-digested DNA) D (digested DNA).
an agarose gel (Fig. 1A). This digestion was done to select those enzymes that could cut at a single site in the genome of the begomovirus generating unit-size molecules, i.e. genome size DNA fragments. The analysis of the gel revealed that some enzymes could generate DNA fragments of the begomovirus genome expected size (ca. 2.6kb, EcoR V and Spe I, arrowhead in Fig. 1A) and others generated smaller DNA fragments (BamH I and Xho I). A band of high molecular weight DNA was still present after digestion with all five enzymes indicating that only part of the amplified DNA was digested, possibly due to the amplification of DNA-A and DNA-B with distinct restriction sites. Hybridization was done with digoxigenin-labeled probes generated by PCR using primers CP1 and CP2 (Nagata et al., in press) for DNA-A and primers CRc2 (Rojas et al., 1993) and B1200F (5 -CCCCTGCAGTAYTAYTGYTGGATGTC-3 , Pst I site undeligned) for DNA-B. PCR was carried out by adding 0.5 l (1 nmol/l) digoxigenin-11-dUTP (Roche) to 100 l of usual PCR reaction. DNA of pCP
(Inoue-Nagata et al., 1999) and of a clone containing the fragment CRc2-B1200F in pGEM-T (Promega) was used as template for PCR for DNA-A and DNA-B probe synthesis, respectively. Hybridization was done according to manufacturer’s instruction (Roche) and visualized by colorimetry. The hybridization with probes specific to the DNA-A and DNA-B confirmed that both components were simultaneously amplified. Hybridization with DNA-A probes (Fig. 1C) showed that only Xho I digested the DNA-A. The absence of the DNA band on the top of the gel indicated that the DNA-A was completely digested generating at least two fragments, one with ca. 2.2 kb and another with ca. 0.5 kb, which was poorly hybridized with DNA-A probe. Hybridization with DNA-B specific probe (Fig. 1B) revealed that EcoR V, Spe I and BamH I digested completely the amplified DNA-B. BamH I digested in at least two sites, while EcoR V and Spe I were most possibly single cutter enzymes. As the aim of this procedure was to clone the complete DNA-B of this virus, the enzyme Spe I was used to digest 10 l of the amplified DNA (3 g). After digestion, the DNA was precipitated and 300 ng were ligated with 50 ng of pBluescript SK+, which was previously digested with Spe I and dephosphorilated according to standard protocols (Sambrook et al., 1989), and transformed into Escherichia coli XL-1 Blue. Twenty-three white colonies were selected; propagated in liquid LB medium, then the plasmid was extracted and digested with Spe I. Out of the 23 clones, 15 showed the expected digestion pattern with DNA fragments of 3 kb (vector) and 2.6 kb. The DNA of two selected clones was column purified (GFX Micro Plasmid Prep Kit, Amersham-Pharmacia) and the insert was sequenced in an automated sequencer (ABI 3100) using the M13 forward and reverse primers. The sequences obtained were examined by the Blast algorithm (http://www.ncbi.nlm.nih.gov/blast/), which demonstrated that the inserted DNA was closely related with the DNA-B of Tomato chlorotic mottle virus (data not shown). Here, we demonstrated that TempliPhi could be efficiently used for cloning of the complete genome of a begomovirus. Direct sequencing of amplified DNA was possible after ethanol precipitation. The use of this method eliminates the inconvenience of the incorporation of error during PCR using non-proofreading enzymes, and potential mutations incorporated by primers. The great advantage of this method was the possibility of cloning from small amounts of viral DNA, a difficult task when using the replicative form procedure. This method was useful to clone the DNA-B of this begomovirus, due to the low concentration of its replicative form in infected tissue.
Acknowledgements We are grateful to Dr. André N. Dusi and Dr. Cláudia S.C. Ribeiro for providing part of the reagents used in this
A.K. Inoue-Nagata et al. / Journal of Virological Methods 116 (2004) 209–211
research. This research was granted by the International Foundation for Science.
References Dellaporta, S.L., Hicks, J.B., 1983. A plant DNA minipreparations. Version II. Plant Mol. Biol. Rep. 1, 19–21. Gilbertson, R.L., Faria, J.C., Hanson, S.F., Morales, F.J., Ahlquist, P., Maxwell, D.P., Russel, D.R., 1991. Cloning of the complete DNA genomes of four bean-infecting geminiviruses and determining their infectivity by electric discharge particle acceleration. Phytopathology 81, 980–985. Inoue-Nagata, A.K., Nagata, T., Miranda, J.D.R., Bezerra, I.C., Ribeiro, S.G., de Ávila, A.C., 1999. Expression of the coat protein of tomato infecting geminivirus in Escherichia coli for serological studies. Virus Rev. Res. 4, 151. Inoue-Nagata, A.K., Nagata, T., Ribeiro, S.G., Bezerra, I.C., Giordano, L.B., de Ávila, A.C., 2001. Comparison of the coat protein cistron of geminiviruses from distinct hosts. Braz. Phytopathol. 26, 527.
211
Nagata, T., Inoue-Nagata, A.K., de Ávila A.C., Giordano, L.B. Print-capture PCR for detection of tomato begomovirus from plants and whiteflies, Braz. Phytopathol., in press. Patel, V.P., Rojas, M.R., Paplomatas, E.J., Gilbertson, R.L., 1993. Cloning biologically active geminivirus DNA using PCR and overlapping primers. Nucleic Acids Res. 11, 1325–1326. Rojas, M.R., Gilbertson, R.L., Russel, D.R., Maxwell, D.P., 1993. Use of degenerate primers in the polymerase chain reaction to detect whitefly-transmited geminiviruses. Plant Dis. 77, 340–347. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning. A Laboratory Manual, second ed. Cold Spring Harbor Laboratory Press, New York. Srivastava, K.M., Hallan, V., Raizada, R.K., Chandra, G., Singh, B.P., Sane, P.V., 1995. Molecular cloning of Indian tomato leaf curl virus genome following a simple method of concentrating the supercoiled replicative from of viral DNA. J. Virol. Methods 51, 297–304. Van Regenmortel, M.H.V., Fauquet, C.M., Bishop, D.H.L., Castens, E., Estes, M.K., Lemon, S., Maniloff, J., Mayo, J.A., McGeoch, D.J., Pring, C.R., Wickner, R. (Eds.), 2000. Virus Taxonomy. Seventh Report of the International Committee on the Taxonomy of Viruses. Academic Press, New York, 1121 pp.
Short communication
A simple method for cloning the complete begomovirus genome using the bacteriophage 29 DNA polymerase Alice K. Inoue-Nagata a,∗ , Leonardo C. Albuquerque a,b , Wesley B. Rocha a,b , Tatsuya Nagata b a
b
Embrapa Hortaliças, Virology, Km. 9, BR060, C. Postal 218, 70359-970 Bras´ılia, DF, Brazil Ciˆencias Biológicas, Universidade Católica de Bras´ılia, W5 Norte, 70790-160 Bras´ılia, DF, Brazil
Received 29 June 2003; received in revised form 13 November 2003; accepted 27 November 2003
Abstract The bacteriophage DNA polymerase amplifies circular DNA in a rolling circle amplification mechanism. This characteristic was applied to amplify and clone the complete circular DNA genome of a begomovirus. Total DNA extracted from infected tissue was used as the template of an amplification reaction using the commercial kit TempliPhi (Amersham Biosciences). The amplified DNA could be used for direct sequencing and was cloned after digestion with a single cutting restriction endonuclease. The use of this enzyme simplified the cloning steps and increased the cloning efficiency of the complete genome of a circular plant DNA virus. © 2003 Elsevier B.V. All rights reserved. Keywords: Circular DNA; Geminiviridae; Rolling circle
Begomoviruses (family Geminiviridae, genus Begomovirus) are circular single-stranded DNA viruses. Members of this genus include monopartite or bipartite viruses (Van Regenmortel et al., 2000). Two methods are used commonly to clone the genome of these viruses. The first method is based on PCR using universal or specific primers, by amplifying either partially or completely the genome (Patel et al., 1993). The second method is based on the extraction of total DNA or semi-purified DNA enriched with the replicative form of the viral genome, followed by digestion with restriction enzymes, Southern blot hybridization and cloning after digestion with a single cutter enzyme (Gilbertson et al., 1991; Srivastava et al., 1995). This procedure usually has low efficiency due to the low concentration of viral genome in the preparation. A simple method is described based on the amplification of the circular DNA using a new commercial kit, TempliPhi (Amersham Biosciences), by a rolling circle amplification using the bacteriophage 29 DNA polymerase. TempliPhi
∗
Corresponding author. Tel.: +55-61-3859067; fax: +55-61-5565744. E-mail address: [email protected] (A.K. Inoue-Nagata).
0166-0934/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2003.11.015
uses an isothermal method for the exponential amplification of circular DNA. The template used for this method was a bipartite begomovirus, DF-BR2, that was found infecting tomato plants in Brazil (Inoue-Nagata et al., 2001). The DNA genome A component (DNA-A) of this virus was already cloned using the traditional method of replicative form isolation. However, the DNA genome B component (DNA-B) could not be successfully cloned, possibly due to the low concentration of this component in the infected tissue. Total DNA was extracted from infected tomato plants (Dellaporta and Hicks, 1983) and subjected to TempliPhi amplification essentially according to the manufacturer’s instructions (Amersham Biosciences). In brief, 5 l of sample buffer and 5 l of reaction buffer was added to 0.5 l of extracted DNA (ca. 0.7 g); to this mixture 0.2 l of enzyme mix was added and incubated at 30 ◦ C for approximately 20 h. Denaturation of the DNA was not essential, since the yield of the amplified DNA fragment did not vary when this step was eliminated (data not shown). The amplified DNA formed a single high molecular weight band in an agarose gel (lane 1, l l of the reaction in Fig. 1A). The DNA (1 l) was digested independently with selected restriction endonucleases and separated in
210
A.K. Inoue-Nagata et al. / Journal of Virological Methods 116 (2004) 209–211
Fig. 1. Agarose gel electrophoresis (0.7%) of amplified DNA digested with restriction enzymes (A), Southern blotting with DNA-B (B) and DNA-A (C) specific probes; 1 kb ladder (Invitrogen) was used as a marker (M). Digestion was done with the following enzymes: non-digested (1), EcoR V (2), Spe I (3), BamH I (4), EcoR I (5) and Xho I (6). Arrowhead in (A) shows unit length DNA; in (B) and (C): ND (non-digested DNA) D (digested DNA).
an agarose gel (Fig. 1A). This digestion was done to select those enzymes that could cut at a single site in the genome of the begomovirus generating unit-size molecules, i.e. genome size DNA fragments. The analysis of the gel revealed that some enzymes could generate DNA fragments of the begomovirus genome expected size (ca. 2.6kb, EcoR V and Spe I, arrowhead in Fig. 1A) and others generated smaller DNA fragments (BamH I and Xho I). A band of high molecular weight DNA was still present after digestion with all five enzymes indicating that only part of the amplified DNA was digested, possibly due to the amplification of DNA-A and DNA-B with distinct restriction sites. Hybridization was done with digoxigenin-labeled probes generated by PCR using primers CP1 and CP2 (Nagata et al., in press) for DNA-A and primers CRc2 (Rojas et al., 1993) and B1200F (5 -CCCCTGCAGTAYTAYTGYTGGATGTC-3 , Pst I site undeligned) for DNA-B. PCR was carried out by adding 0.5 l (1 nmol/l) digoxigenin-11-dUTP (Roche) to 100 l of usual PCR reaction. DNA of pCP
(Inoue-Nagata et al., 1999) and of a clone containing the fragment CRc2-B1200F in pGEM-T (Promega) was used as template for PCR for DNA-A and DNA-B probe synthesis, respectively. Hybridization was done according to manufacturer’s instruction (Roche) and visualized by colorimetry. The hybridization with probes specific to the DNA-A and DNA-B confirmed that both components were simultaneously amplified. Hybridization with DNA-A probes (Fig. 1C) showed that only Xho I digested the DNA-A. The absence of the DNA band on the top of the gel indicated that the DNA-A was completely digested generating at least two fragments, one with ca. 2.2 kb and another with ca. 0.5 kb, which was poorly hybridized with DNA-A probe. Hybridization with DNA-B specific probe (Fig. 1B) revealed that EcoR V, Spe I and BamH I digested completely the amplified DNA-B. BamH I digested in at least two sites, while EcoR V and Spe I were most possibly single cutter enzymes. As the aim of this procedure was to clone the complete DNA-B of this virus, the enzyme Spe I was used to digest 10 l of the amplified DNA (3 g). After digestion, the DNA was precipitated and 300 ng were ligated with 50 ng of pBluescript SK+, which was previously digested with Spe I and dephosphorilated according to standard protocols (Sambrook et al., 1989), and transformed into Escherichia coli XL-1 Blue. Twenty-three white colonies were selected; propagated in liquid LB medium, then the plasmid was extracted and digested with Spe I. Out of the 23 clones, 15 showed the expected digestion pattern with DNA fragments of 3 kb (vector) and 2.6 kb. The DNA of two selected clones was column purified (GFX Micro Plasmid Prep Kit, Amersham-Pharmacia) and the insert was sequenced in an automated sequencer (ABI 3100) using the M13 forward and reverse primers. The sequences obtained were examined by the Blast algorithm (http://www.ncbi.nlm.nih.gov/blast/), which demonstrated that the inserted DNA was closely related with the DNA-B of Tomato chlorotic mottle virus (data not shown). Here, we demonstrated that TempliPhi could be efficiently used for cloning of the complete genome of a begomovirus. Direct sequencing of amplified DNA was possible after ethanol precipitation. The use of this method eliminates the inconvenience of the incorporation of error during PCR using non-proofreading enzymes, and potential mutations incorporated by primers. The great advantage of this method was the possibility of cloning from small amounts of viral DNA, a difficult task when using the replicative form procedure. This method was useful to clone the DNA-B of this begomovirus, due to the low concentration of its replicative form in infected tissue.
Acknowledgements We are grateful to Dr. André N. Dusi and Dr. Cláudia S.C. Ribeiro for providing part of the reagents used in this
A.K. Inoue-Nagata et al. / Journal of Virological Methods 116 (2004) 209–211
research. This research was granted by the International Foundation for Science.
References Dellaporta, S.L., Hicks, J.B., 1983. A plant DNA minipreparations. Version II. Plant Mol. Biol. Rep. 1, 19–21. Gilbertson, R.L., Faria, J.C., Hanson, S.F., Morales, F.J., Ahlquist, P., Maxwell, D.P., Russel, D.R., 1991. Cloning of the complete DNA genomes of four bean-infecting geminiviruses and determining their infectivity by electric discharge particle acceleration. Phytopathology 81, 980–985. Inoue-Nagata, A.K., Nagata, T., Miranda, J.D.R., Bezerra, I.C., Ribeiro, S.G., de Ávila, A.C., 1999. Expression of the coat protein of tomato infecting geminivirus in Escherichia coli for serological studies. Virus Rev. Res. 4, 151. Inoue-Nagata, A.K., Nagata, T., Ribeiro, S.G., Bezerra, I.C., Giordano, L.B., de Ávila, A.C., 2001. Comparison of the coat protein cistron of geminiviruses from distinct hosts. Braz. Phytopathol. 26, 527.
211
Nagata, T., Inoue-Nagata, A.K., de Ávila A.C., Giordano, L.B. Print-capture PCR for detection of tomato begomovirus from plants and whiteflies, Braz. Phytopathol., in press. Patel, V.P., Rojas, M.R., Paplomatas, E.J., Gilbertson, R.L., 1993. Cloning biologically active geminivirus DNA using PCR and overlapping primers. Nucleic Acids Res. 11, 1325–1326. Rojas, M.R., Gilbertson, R.L., Russel, D.R., Maxwell, D.P., 1993. Use of degenerate primers in the polymerase chain reaction to detect whitefly-transmited geminiviruses. Plant Dis. 77, 340–347. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning. A Laboratory Manual, second ed. Cold Spring Harbor Laboratory Press, New York. Srivastava, K.M., Hallan, V., Raizada, R.K., Chandra, G., Singh, B.P., Sane, P.V., 1995. Molecular cloning of Indian tomato leaf curl virus genome following a simple method of concentrating the supercoiled replicative from of viral DNA. J. Virol. Methods 51, 297–304. Van Regenmortel, M.H.V., Fauquet, C.M., Bishop, D.H.L., Castens, E., Estes, M.K., Lemon, S., Maniloff, J., Mayo, J.A., McGeoch, D.J., Pring, C.R., Wickner, R. (Eds.), 2000. Virus Taxonomy. Seventh Report of the International Committee on the Taxonomy of Viruses. Academic Press, New York, 1121 pp.