Determination of gene copy number and genotype of transgenic Arabidopsis thaliana by competitive PCR.
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.
Honda
Munechika
M
Laboratory of Plant Biotechnology and Microbiology, Gifu Prefectural Institute for Bio-industrial Technology, 3481-2 Kamihachiya, Hachiya-cho, Minokamo, Gifu, 505-0004, Japan. honda@bio.rd.pref.gifu.jp
Muramoto
Yasunori
Y
Kuzuguchi
Tsuyoshi
T
Sawano
Sadanori
S
Machida
Masayuki
M
Koyama
Hiroyuki
H
D015111:4-Hydroxyphenylpyruvate Dioxygenase / Q000235:genetics; D017360:Arabidopsis / Q000235:genetics*; D015139:Blotting, Southern; D002950:Citrate (si)-Synthase / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D018628:Gene Dosage; D005838:Genotype; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction / Q000379:methods*
D004269:DNA, Bacterial; D018744:DNA, Plant; C036483:T-DNA; D015111:4-Hydroxyphenylpyruvate Dioxygenase; D002950:Citrate (si)-Synthase