Agronomy and Horticulture Department
Real Time PCR - Some Basic Principles
Date of this Version
2004
Document Type
Article
Citation
Plant and Soil Sciences eLibrary (PASSeL) Lesson
A contribution of the University of Nebraska College of Agricultural Sciences and Natural Resources, Lincoln Nebraska 68583, Journal Series No. 04-13
Abstract
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 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.
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:
- 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.
Modules:
- Lesson home
- Real Time PCR - Some Basic Principles Introduction
- Need for Detecting GMOs?
- GMO Detection
- Presence/Absence Not Enough
- Defining Real Time PCR
- Temperature Cycles in Real Time PCR
- Real Time PCR Components
- Probe Systems - SYBR Green
- Probe Systems - FRET
- Probe Systems - Taqman
- Using Data from Real Time PCR
- Strengths/weaknesses of Real Time PCR
- Summary for Real Time PCR - Basic Principles
- References for Real Time PCR - Basic Principles
- Glossary
- Videos
Comments
Copyright © 2004 Deana M. Namuth and G. Ronald Jenkins. Used by permission.
JNRLSE approved 2005
This project was supported in part by the National Research Initiative Competitive Grants CAP project 2011-68002-30029 from the USDA National Institute of Food and Agriculture, administered by the University of California-Davis and by the National Science Foundation (NSF), Division of Undergraduate Education, National SMETE Digital Library Program, Award #0938034, administered by the University of Nebraska. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of the USDA or NSF.
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 authors and do not necessarily reflect the view of the U.S. Department of Agriculture.