Identification of Gene Copy Number in the Transgenic Plants by Quantitative Polymerase Chain Reaction (qPCR)

Transgenic events are defined as exogenous DNA insertion in the genome through genetic transformation. It is a powerful means for the improvement of crop plants and to understand the gene function. Multiple DNA insertion events may occur at one or several chromosomal locations. One of the important tasks, after validation of the transformation of transgenic plants, is the identification of single copy in the transgenic. This means the insertion of exogenous DNA fragment only in a single locus in the genome. Southern blot hybridization is a convincing and reliable method, for estimation of copy number in transgenic lines but it is cumbersome and time-consuming process. One of the other well-known methods is quantitative polymerase chain reactions (qPCR), a simple and rapid method to identify copy number from a population of independent transgenic lines. In comparison to the Southern hybridization method, qPCR is simpler to perform, requires less DNA, lesser time and does not require any labeled probes. This method utilizes specific primers to amplify target transgenes and endogenous reference genes. Designing an appropriate and specific primer pair is a very crucial part of the estimation of the gene copy number. In this chapter, we have illustrated a detailed methodology for identification of the gene copy of the transgenic plants.