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Real Time PCR - Some Basic Principles

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Strengths/weaknesses of Real Time PCR

Real time PCR has advantages over nonconventinal PCR because it provides a wide dynamic range for quantification, contains high sensitivity, and improved precision. Also, since the method is being performed in a real-time format, no post-PCR steps are required, such as agarose gel electrophoresis. Real time PCR has the ability to detect very small amounts of target DNA, but that also means small experimental errors are also detected and amplified, increasing the risk of unreliable results. The accuracy of data, using real time PCR, is largely dependent upon several factors within the sample itself, as well as those in the laboratory. Even the randomness of a sample selection (i.e. 1000 seed count from a truckload) will impact the results of accuracy (Jenkins, 2002). From the sample perspective, knowing the zygosity is important. For example corn leaf tissue is 2n, but endosperm is 3n. The parental breeding lines might be homozygous, but the variety a producer grows might be hemizygous. Then to make things even more complicated, the number of gene copies actually inserted during the plant transformation process (i.e. gene gun, Agrobacterium) will vary from one to many copies per plant genome. Reference materials where these characteristics are defined can be used as controls to standardize the tests.

Figure 21. USDA-GIPSA scientist pipetting PCR reagents.


Several laboratory factors affect real time PCR data results as well. A few are described here. Researchers have found that chemicals naturally present in plant tissue, such as glucuronic acid and alginic acid can interfere with the success of real time PCR (Figure 21). Also, certain combinations of PCR reagents perform better than others (Holden et al., 2003). Even the type of commercially available Taq polymerase or an individual PCR machine being used can give varying effects. However, the use of endogenous gene controls aid in finely tuning data obtained in the real time PCR assay. Successful amplification of an endogenous gene is not a guarantee that the DNA is of sufficient purity for amplification and detection of a gene present in many fewer copies, such as would be expected in export shipments containing some small percentage of GMO’s (Holden et al., 2003).

 As these variable examples indicate, strict and careful laboratory procedures must be followed in order to produce reliable and reproducible results. The USDA-GIPSA (United States Department of Agriculture - Grain Inspection, Packers and Stockyards Administration division) is an organization that has established a proficiency program for improving the reliability and credibility of PCR based testing for biotechnology-derived events and the development of reference materials (Figure 22).

Figure 22. USDA-GIPSA scientist testing materials in the proficiency program.

The proficiency program has proven to be beneficial for organizations that test for the presence of biotechnology-derived events in grains and oil seeds, with many organizations from several countries enrolled in the program.

The National Institute of Standards and Technology (NIST) (http://www.cstl.nist.gov/div831/) and the USDA/GIPSA (http://www.gipsa.usda.gov/fgis/standproc/biotech.html) have established confidentiality and/or material transfer agreements with life science organizations and industries that develop biotechnology-derived events in grains and oilseeds. With these agreements in place, NIST and USDA/GIPSA have created an ongoing collaborative research program to improve DNA-based testing methods and investigate the development of reference materials. This will make possible inter-laboratory calibration and validation of methods in real time PCR assays.

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