Real Time PCR—Some Basic Principles

Real time PCR is a laboratory technique that can perform relatively accurate, reliable, and reproducible measurements to quantitatively determine the presence of specific gene sequences. Its value is being recognized in a variety of plant science applications, including transgenic (GMO) detection. It is becoming increasingly important to know what percentage of a particular transgene is present in an export shipment, for example. Real time PCR can also be used to support more traditional plant breeding techniques, making the process of distinguishing allelic variations more efficient. This lesson explains the principles of real time PCR and its application in plant breeding and GMO detection. This is a lesson in a series found in the Library of Crop Technology (http://croptechnology.unl.edu).This lesson assumes you are familiar with conventional PCR methods and builds upon those principles. If, however, you need some background information on conventional PCR, please refer to the Polymerase Chain Reaction lesson (http://croptechnology.unl.edu/viewLesson.cgi?LessonID=968252315). The objectives of this lesson are as follows: Describe how a GMO can be differentiated from a non‐GMO. Define real time PCR and contrast it with the conventional PCR method. Identify and contrast different probe detection systems. Explain, in detail, the Taqman system. Analyze the overall strengths and weaknesses of real time PCR. A bank of quiz questions focused on these objectives is a part of this lesson. The lesson is written to target the educational needs of graduate students and advanced extension audiences.