title: Rapid determination of transgene copy number in tobacco by competitive PCR using a pair of SSR primers.
abstract: We developed a straightforward, rapid, and inexpensive method to determine transgene copy number in tobacco. The plasmid (pSSRCopy) used for tobacco transformation contains a simple sequence repeat (SSR) locus, PT1199, which was partially deleted in the middle, a homogenous SSR locus in tobacco K326. A 168-bp segment of the cloned PT1199 was shortened to 95 bp by deleting a 73-bp internal fragment. Using a pair of SSR primers, competitive PCR was amplified from genomic DNA from transgenic tobacco harboring pSSRCopy, and the two expected bands were found. The 168-bp band (SSR-168) corresponds to endogenous PT1199 and the 95-bp band (SSR-95) comes from the integrated pSSRCopy. A single copy of a transgene can be easily distinguished from multiple copies by comparing band densities.
authors.affiliation: College of Horticulture, Nanjing Agricultural University, Nanjing, Jiangsu, China.
authors.forename: K Y
authors.initials: KY
authors.lastname: Xu
authors.forename: S H
authors.initials: SH
authors.lastname: Wang
authors.forename: L
authors.initials: L
authors.lastname: Xi
authors.forename: Q J
authors.initials: QJ
authors.lastname: Wang
authors.forename: C
authors.initials: C
authors.lastname: Dong
authors.forename: J Y
authors.initials: JY
authors.lastname: Zhang
authors.forename: S C
authors.initials: SC
authors.lastname: Qu
authors.forename: Z
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D015139:Blotting, Southern; D017931:DNA Primers / Q000235:genetics*; D018628:Gene Dosage; D017343:Genes, Plant; D005821:Genetic Techniques; D008957:Models, Genetic; D018515:Plant Leaves / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D014026:Nicotiana / Q000235:genetics*; D019076:Transgenes
chemical_list: D017931:DNA Primers