Quantitative Real-Time PCR Assay for Determining Transgene Copy Number in Transformed Plants

Abstract

The development of transgenic events can be limited by many factors. These include expression levels, insert stability and inheritance, and the identification of simple insertion events. All of the factors can be related to the copy number of the transgene. Traditionally, copy number has been determined by laborious blotting techniques. We have developed an alternative approach that utilizes the fluorogenic 5′ nuclease (TaqMan®) assay to quantitatively determine transgene copy level in plants. Using this assay, hundreds of samples can be analyzed per day in contrast to the low throughput encountered with traditional methods. To develop the TaqMan copy number assay, we chose to utilize our highly efficient Agrobacterium-mediated transformation system of maize. This transformation procedure generates predominantly low copy number insertion events, which simplified assay development. We have also successful applied this assay to other crops and transformation systems.