Summary for Real Time PCR - Basic Principles
As we have seen in this lesson, there are several needs for gaining information on specific DNA sequences a crop plant may contain. Scientists, plant breeders, government regulators and consumers are in situations where it is important to know not only if a transgene is present, but also how many copies of such a gene may be present. Real time PCR has been widely used to quantitatively estimate such measurements. It can detect very small amounts of target DNA, with high sensitivity, relatively good accuracy and improved precision.
Figure 23: A centrifugation step in the real time PCR process.
Real time PCR can utilize several different probe chemistries, where fluorescence emission is detected as the PCR reactions take place rather than at the completion of all cycles. The SYBR Green probe system intercalates into double stranded DNA molecules, while Taqman and FRET utilize primers and labeled probes to be more sequence specific. The amount of fluorescence emitted is proportional to the amount of the particular DNA sequence being studied, such as a transgene.
The accuracy of data, using real time PCR, is largely dependent upon several factors including sample preparation, quality performance of standard reference materials, and choice of an endogenous control gene. The USDA-GIPSA Proficiency Program is an example of collaborations formed to make steps towards standardizing the use of real time PCR in GMO detection.