K.Y. Xu, S.H. Wang, L. Xi, Q.J. Wang, C. Dong, J.Y. Zhang, S.C. Qu and Z. Zhang
Published May 18, 2010
Genet. Mol. Res. 9 (2): 935-940 (2010)
DOI 10.4238/vol9-2gmr790
About the authors
K.Y. Xu, S.H. Wang, L. Xi, Q.J. Wang, C. Dong, J.Y. Zhang, S.C. Qu and Z. Zhang
Corresponding author
Z. Zhang
E-mail: zhangzh@njau.edu.cn / qscnj@njau.edu.cn
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 singlecopy of a transgene can be easily distinguished from multiple copies by comparing band densities.
Key words: Copy number; Transgene; Competitive PCR; SSR; Tobacco