Abstract
Multiplex PCR is a variant of conventional PCR which includes two or more pairs of primers in a single reaction to amplify corresponding genes simultaneously. In this study, a reliable multiplex PCR analysis protocol was established for simple and fast detection of transgenes in plant materials. Two pairs of primers, corresponding to neomycin phosphotransferase gene and 1-aminocyclopropane-1-carboxylate synthase gene, were selected for target and resident gene respectively. The method bypasses routine DNA extraction, requires only very little amount of plant tissue and produces reliable results as shown by successful discrimination of transformed and nontransformed tobacco, tomato and kumquat materials. The method facilitates early identification of transgenic buds when they are still quite small.
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Abbreviations
- ACS:
-
1-aminocyclopropane-1-carboxylate synthase
- ACSDP:
-
ACS downstream primer
- ACSUP:
-
ACS upstream primer
- MPCR:
-
multiplex polymerase chain reaction
- NPT:
-
neomycin phosphotransferase
- NPTDP:
-
NPT downstream primer
- NPTUP:
-
NPT upstream primer
References
Berthomieu P., Meyer C. 1991. Direct amplification of plant genomic DNA from leaf and root pieces using PCR. Plant Mol. Biol., 17: 555–557.
Chamberlain J. S., Gibbs R. A., Ranier J. E., Nguyen P. N., Caskey C. T. 1988. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucl. Acid. Res., 16: 11141–11156.
Edwards K., Johnstone C., Thompson C. 1991. A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucl. Acid. Res., 1991, 19: 1349.
Henegariu O., Heerema N., Dlouhy S. R., Vance G., Vogt P. H. 1997. Multiplex PCR: Critical parameters and step-by-step protocol. Bio Techniques, 23: 504–511.
Hull G. A., Devic M. 1995. The β-glucuronidase (gus) reporter gene system. Meth. Mol. Biol., 49: 125–141.
James D., Schmidt A. M., Wall E., Green M., Masri S. 2003. Reliable detection and identification of genetically modified maize, soybean, and canola by multiplex PCR analysis. J. Agric. Food Chem., 51: 5829–5834.
Klimyuk V. I., Carroll B. J., Thomas C. M., Jones J. D. G. 1993. Alkali treatment for rapid preparation of plant material for reliable PCR analysis. Plant J., 3: 493–494.
Mannerlöf M., Tenning P. 1997. Screening of transgenic plants by multiplex PCR. Plant Mol. Biol. Rep., 15: 38–45.
McCarthy P. L., Hansen J. L., Zemetra R. S., Berger P. H. 2002. Rapid identification of transformed wheat using a half-seed PCR assay. BioTechniques, 32: 560–564.
Permingeat H. R., Reggiardo M. I., Vallejos R. H. 2002. Detection and quantification of transgenes in grains by multiplex and real-time PCR. J. Agric. Food Chem., 50: 4431–4436.
Sambrook J., Russell D. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Habor, New York: 8.5.
Tao Z., Cai X. F., Yang S. L., Gong Y. 2001. Detection of exogenous genes in genetically modified plants with multiplex polymerase chain reaction. Plant Mol. Biol. Rep., 19: 289–298.
Tengel C., Schüßler P., Balles J., Sprenger-Haußels M. 2001. PCR-based detection of genetically modified soybean and maize in raw and highly processed food-stuffs. BioTechniques, 31: 426–429.
Xu C. J., Chen K. S., Zhang S. L. 2001. Molecular cloning of four members of ACC synthase gene family from kiwifruit (Actinidia chinensis Planch.). J. Agric. Biotechnol., 9: 55–57
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Xu, CJ., Yang, L. & Chen, KS. Development of a rapid, reliable and simple multiplex PCR assay for early detection of transgenic plant materials. Acta Physiol Plant 27, 283–288 (2005). https://doi.org/10.1007/s11738-005-0004-8
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DOI: https://doi.org/10.1007/s11738-005-0004-8