title: Standard addition quantitative real-time PCR (SAQPCR): a novel approach for determination of transgene copy number avoiding PCR efficiency estimation.
abstract: Quantitative real-time polymerase chain reaction (qPCR) has been previously applied to estimate transgene copy number in transgenic plants. However, the results can be erroneous owing to inaccurate estimation of PCR efficiency. Here, a novel qPCR approach, named standard addition qPCR (SAQPCR), was devised to accurately determine transgene copy number without the necessity of obtaining PCR efficiency data. The procedures and the mathematical basis for the approach are described. A recombinant plasmid harboring both the internal reference gene and the integrated target gene was constructed to serve as the standard DNA. It was found that addition of suitable amounts of standard DNA to test samples did not affect PCR efficiency, and the guidance for selection of suitable cycle numbers for analysis was established. Samples from six individual T(0) tomato (Solanum lycopersicum) plants were analyzed by SAQPCR, and the results confirmed by Southern blot analysis. The approach produced accurate results and required only small amounts of plant tissue. It can be generally applied to analysis of different plants and transgenes. In addition, it can also be applied to zygosity analysis.
authors: Laboratory of Fruit Quality Biology, Zhejiang University, Hangzhou, PR China.
authors: Yuji
authors: Y
authors: Huang
authors: Xueren
authors: X
authors: Yin
authors: Changqing
authors: C
authors: Zhu
authors: Weiwei
authors: W
authors: Wang
authors: Donald
authors: D
authors: Grierson
authors: Changjie
authors: C
authors: Xu
authors: Kunsong
authors: K
authors: Chen
mesh_terms: D015139:Blotting, Southern; D004247:DNA / Q000235:genetics; D018628:Gene Dosage; D018551:Solanum lycopersicum / Q000235:genetics*; D030821:Plants, Genetically Modified; D010957:Plasmids; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D012015:Reference Standards; D019076:Transgenes
chemical_list: D004247:DNA