Location: Corn Insects and Crop Genetics ResearchTitle: Determination of Transgene Copy Number by Real-time Quantitative-PCR) Author
Submitted to: Book Chapter
Publication Type: Book / chapter
Publication Acceptance Date: 8/21/2008
Publication Date: 4/1/2009
Citation: Shepherd, C.T., Moran Lauter, A., Scott, M.P. 2009. Determination of Transgene Copy Number by Real-time Quantitative-PCR. In: Scott, M.P, editor. Transgenic Maize. Totowa, NJ: Humana Press. p. 23-28. Interpretive Summary: Transgenic plants are produced for both basic research and commercial purposes. The methods used to generate transgenic plants are not specific in the number of copies of a transgene that are incorporated into the plant’s genome. Transgene copy number directly relates to the stability, inheritance and expression level of the transgene. Determining copy number is an important step in transformant characterization and can differentiate between complex (multiple transgenes) and simple (single transgene) transformation events. Traditional methods to determine copy number are time consuming and difficult to perform on a large scale, making it inefficient to use when analyzing large numbers of transgenic plants. They also often involve the use of radioactive materials. We describe a method utilizing real-time quantitative polymerase chain reaction (RT-qPCR), a relatively new technology. RT-qPCR reactions take hours to run and yield data in digital form for analysis, as opposed to days of work using traditional methods. Since this technique is non-radioactive, less training is required for the user. Researchers in both the private and public sectors can use our method to increase efficiency in characterizing large numbers of transgenic plants.
Technical Abstract: Efficient methods to characterize transgenic plants are important to quickly understand the state of the transformant. Determining transgene copy number is an important step in transformant characterization and can differentiate between complex and simple transformation events. This knowledge can be extremely useful when determining what future experiments and uses the transgenic lines can be utilized for. The method described here uses real-time quantitative PCR to determine the transgene copy number present in the genome of the transformant. Specifically, this method measures the relative transgene copy number by comparing it with an endogenous gene with a known copy number. This method is a quick alternative to the Southern blot, a method that is commonly used to determine gene copy number, and is effective when screening large numbers of transformants.