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Journal of Experimental Botany, Vol. 53, No. 373, pp. 1515-1520, June 2002
© 2002 Oxford University Press

Determination of gene copy number and genotype of transgenic Arabidopsis thaliana by competitive PCR

Munechika Honda1,4, Yasunori Muramoto1, Tsuyoshi Kuzuguchi1, Sadanori Sawano1, Masayuki Machida2 and Hiroyuki Koyama3

1 Laboratory of Plant Biotechnology and Microbiology, Gifu Prefectural Institute for Bio-industrial Technology, 3481-2 Kamihachiya, Hachiya-cho, Minokamo, Gifu, 505-0004, Japan
2 Institute of Molecular and Cell Biology, National Institute of Advanced Industrial Science and Technology, 1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan
3 Laboratory of Plant Cell Technology, Faculty of Agriculture, Gifu University, 1–1 Yanagido, Gifu, 501-1193, Japan

A simple and rapid method is described for determining the integrated T-DNA copy number and the genotype in transgenic Arabidopsis thaliana by two-step competitive PCR. First, the amount of genomic DNA in the extracts, obtained from an individual A. thaliana transformant, was accurately determined by the 1st competitive PCR using a known single copy gene, 4HPPD (4-hydroxyphenylpyruvate dioxygenase), as a target. Second, the number of T-DNA copies per genome was estimated by quantifying the NPTII gene, which was involved in the T-DNA, by the 2nd competitive PCR using exactly the same amount of genomic DNA for each sample. The estimated copy number and genotype obtained by this procedure were identical to those determined by Southern blot analysis and segregation analysis.

Key words: Arabidopsis thaliana, gene copy number, genotype, quantitative PCR.


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