Rapid determination of transgene copy number in tobacco by competitive PCR using a pair of SSR primers.
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.
Xu
K Y
KY
College of Horticulture, Nanjing Agricultural University, Nanjing, Jiangsu, China.
Wang
S H
SH
Xi
L
L
Wang
Q J
QJ
Dong
C
C
Zhang
J Y
JY
Qu
S C
SC
Zhang
Z
Z
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
D017931:DNA Primers