Abstract
We describe the simultaneous amplification of different segments of foreign DNA in transgenic plants using the polymerase chain reaction (PCR). We used PCR to simultaneously amplify different regions of transformed T-DNA in order to assay the integrity of transformed constructions in primary tomato transformants. We also used simultaneous PCR amplification to examine the segregation of transformed sequences in progeny of primary transformants. A tomato transformant containing the maize transposable elementAc was crossed to transformants containing the non-autonomousDs1 element flanked by maizeAdh1 sequences. We then ran PCR reactions on DNA from F1 progeny using two sets of primers, one set homologous toAc and one set homologous toAdh1 sequences on either side ofDs1. Because theAc andAdh1 primers resulted in amplification of fragments of different sizes, it was possible to monitor the inheritance ofAc and theDs1 containingAdh1 genein a single reaction. Additionally, it was possible to identify F1 plants in whichDs1 had excised by the amplification of a fragment the size predicted for an empty donor site. In order to run these reactions, we have constructed a simple and inexpensive thermal cycler which, when used in conjunction with the rapid miniscreen plant DNA isolation procedure described, allows the processing of a large number of samples in a single day. Therefore, we have shown that PCR can be a useful tool to monitor the integrity of foreign genes in transgenic plants, to follow the segregation of foreign DNA in progeny, and to assay for the excision of transposable elements.
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Abbreviations
- PCR:
-
polymerase chain reaction
- CTAB:
-
hexadecyltrimethyl-ammonium bromide
- TE:
-
10 mM Tris, HCl, 1 mM EDTA, pH 8
- EEO:
-
electroendosmosis
References
Baker, B., Schell, J., Lörz, H., and Fedoroff, N. 1986. Transposition of the maize controlling element “Activator” in tobacco. Proc. Nat. Acad. Sci USA. 83:4844–4848.
Bernatzky, R., and Tanksley, S.D. 1986. Genetics of actin-related sequences in tomato. Theor. Appl. Genet. 72:314–321.
Clarke, B.C., Moran, L.B., and Appel, R. 1989. DNA analyses in wheat breeding. Genome, in press.
Deroles, S.C., and Gardner, R.C. 1988. Analysis of the T-DNA structure in a large number of transgenic petunias generated byAgrobacterium-mediated transformation. Plant Mol. Biol. 11:365–377.
Fraley, R.T., Rogers, S.G., Horsch, R.B., Eichholtz, D.A., Flick, J.S., Fink, C.L., Hoffman, N.L., and Sanders, P.R. 1985. The SEV system: a new disarmed Ti plasmid vector for plant transformation. Biotechnology 3:629–635.
Frohman, M.A., Dush, M.K., and Martin, G.R. 1988. Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85:8998–9002.
Innis, M.A., Myambo, K.B., Gelfand, D.H., and Brow M.A.D. 1988. DNA sequencing withThermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc. Natl. Acad. Sci. USA 85:9436–9440
Jones, J.D.G., Gilbert, D.E., Grady, K.L., and Jorgensen, R.A. 1987. T-DNA structure and gene expression in petunia plants transformed byAgrobacterium tumefaciens derivatives. Mol. Gen. Genet. 207:478–485.
Jorgenson, R., Snyder, C., and Jones, J.D.G. 1987. T-DNA is organized predominantly in inverted repeat structures in plants transformed withAgrobacterium tumefaciens C58 derivatives. Mol. Gen. Genet. 207:471–477.
Kawasaki, E.S., Clark, S.S., Coyne, M.Y., Smith, S.D., Champlin, R., Wite, O.N., and McCormick, F.P. 1988. Diagnosis of chronic myeloid and acute lymphocytic leukemias by detection of leukemia-specific mRNA sequences amplifiedin vitro. Proc. Natl. Acad. Sci. USA 85:5698–5702.
Knapp, S., Coupland, G., Uhrig, U., Starlinger, P., and Salamini, F. 1988. Transposition of the maize transposable elementAc inSolanum tuberosum. Mol. Gen. Genet. 213:285–290.
Lassner, M.W., Palys, J.M., and Yoder, J.I. 1989. Genetic transactivation ofDissociation elements in transgenic tomato plants. Mol. Gen. Genet., in press.
Lee, M.S., Chang, K.S., Cabanillas, F., Freireich, E.J., Trujillo J.M., and Stass, S.A. 1987. Detection of minimal residual cells carrying the t(14;18) by DNA sequence amplification. Science 237:175–178.
Loh, E.Y., Elliot, J.F., Cwirla, S., Lanier, L.L., and Davis, M.M. 1989. Polymerase chain reaction with single-sided specificity: analysis of T cell receptor d chain. Science 243:217–220.
Müller-Neumann, M., Yoder, J.I., and Starlinger, P. 1984. The DNA sequence of transposable elementAc ofZea mays L. Mol. Gen. Genet. 198:19–24.
Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A., and Arnheim, N. 1985. Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350–1354.
Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., Erlich, H.A. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491.
Scharf, S.J., Horn, G.T., and Erlich, H.A. 1986. Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science 233:1076–1078.
Schell, J.S. 1987. Transgenic plants as tools to study the molecular organization of plant genes. Science 237:1176–1182.
Spielmann, A., and Simpson R.B. 1986. T-DNA structure in transgenic tobacco plants with multiple independent integration sites. Mol. Gen. Genet. 205:34–41.
Sutton W.D., Gerlach, W.L., Schwartz, D., and Peacock, W.J. 1984. Molecular analysis ofDs controlling element mutations at theAdh locus of maize. Science 223:1265–1268.
Wong, C., Dowling, C.E., Saiki, R.K., Higuchi, R.G., Erlich, H.A., and Kazazian, H.H. 1987. characterization of β-thalassaemia mutations using direct genomic sequencing of amplified single copy DNA. Nature 330:384–386.
Yoder, J.I., Palys, J., Alpert, K., and Lassner, M. 1988.Ac transposition in transgenic tomato plants. Mol. Gen. Genet. 213:291–296.
Van Sluys, M.A., Tempé, J., and Fedoroff, N. 1987. Studies on the introduction and mobility of the maizeActivator element inArabidopsis thaliana andDaucus carota. EMBO J. 6:3881–3889.
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Lassner, M.W., Peterson, P. & Yoder, J.I. Simultaneous amplification of multiple DNA fragments by polymerase chain reaction in the analysis of transgenic plants and their progeny. Plant Mol Biol Rep 7, 116–128 (1989). https://doi.org/10.1007/BF02669627
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DOI: https://doi.org/10.1007/BF02669627