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Real Time PCR - Some Basic Principles

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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 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, with examples in plant breeding and GMO detection.

Real Time PCR - Some Basic Principles Introduction

Deana M. Namuth
Department of Agronomy and Horticulture
University of Nebraska-Lincoln, USA

(http://agronomy.unl.edu/namuth-covert)
G. Ronald Jenkins
United States Department of Agriculture
Grain Inspection, Packers and Stockyards Administration

(http://www.usda.gov/gipsa/)

2004

Peer Reviewed Web Lesson JNRLSE Approved 2005

Lesson Navigation Tips:
- Click on ’Animations’ button found to the left in order to view the animation which supplements this lesson. You can also click on the animation icon within the text.
- Click once on figures to see enlarged versions.
- Click once on words in color to bring up their definitions.

Overview 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.

Objectives

 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.
At the completion of this lesson, learners will be able to:

1. Describe how a GMO can be differentiated from a non-GMO.
2. Define real time PCR and contrast it with the conventional PCR method.
3. Identify and contrast different probe detection systems.
4. Explain in detail, the Taqman system.
5. Analyze the overall strengths and weaknesses of real time PCR.  


Development of this lesson was supported in part by USDA Initiative for Future Agriculture and Food Systems (IFAFS)
and the Cooperative State Research, Education, & Extension Service,
U.S. Dept of Agriculture under Agreement Number 00-52100-9710.
Any opinions, findings, conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture.

A contribution of the University of Nebraska College of Agricultural Sciences and Natural Resources, Lincoln Nebraska 68583, Journal Series No. 04-13

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