Using Data from Real Time PCR

With conventional PCR a sample of the final reactions is taken from each tube and run through a gel electrophoresis to separate DNA bands (Electrophoresis: How scientists observe fragments of DNA). The gel is stained and these bands, containing the amplified (copied) DNA, are visible under ultra-violet light (Figure 17).

Figure 17. DNA visualized in a gel placed onto an ultra-violet light source.

However, this visualization is not quantitative, instead merely detects the presence of a certain DNA sequence. The number of DNA copies making the bands is not calculated nor is the amount of a specific gene present in the starting sample measurable. In real time PCR though, measurements are detected at each cycle and the data collected by a computer. The resulting graph of fluorescence measurements at each cycle is used to define the exponential phase of the reaction, a prerequisite for accurate determination of copy number at the beginning of the reaction.

The image shown in Figure 18 graphically illustrates the differences in observing the results of conventional and real time PCR. The arrows show where fluorescence in a Taqman system is first detected at a significant level for each sample being tested for a Bt event. We can easily determine that each sample contains a different concentration level of Bt genes at this point of the real time PCR experiment.

The first arrow on the left shows the sample which had a significant fluorescence detected earliest. Therefore it has more copies of the Bt event present initially. If we were using conventional PCR, our detection measurement would not take place until the end of the experiment, as indicated by the circle in this figure. Due to the exponential amplification nature of PCR, by the end of the experiment, it is impossible to discern differences in Bt gene concentrations among the samples.

Figure 18. Graph showing detection differences between Real Time and Conventional PCR measurements

The image shown in Figure 19 is obtained from a real time PCR experiment. The cycle numbers are along the x-axis and amount of fluorescence along the y-axis. In this experiment GMO-DNA standards at 10%, 1%, 0.1% and 0.01 % GMO DNA, along with an unknown sample were tested. At approximately cycle #24 the fluorescence peak for the 10% GMO standard crosses the CT threshold (red line) and goes into the exponential phase. Signals detected below the CT are merely background noise. The least concentrated GMO-DNA standard crosses the CT threshold at Cycle 31. Therefore, the more copies of a gene present initially, the quicker the amount of fluorescence emitted will reach the CT threshold.

Figure 19. Data from a real time PCR experiment with known varying concentrations of a gene standard.

Figure 20. Plotting real time PCR data from a sample with unknown gene concentration onto the standards graph from the same experiment.

In Figure 20 the unknown sample fluorescence data is applied and falls along the 0.1% GMO-standard graph. This indicates that the unknown sample contains the same amount of GMO-DNA as the 0.1% concentration standard.

Similarly, genotype differences can be measured. For example in a plant breeding program, if a Taqman probe is designed to bind to a 'R' gene allele but not to a 'r' allele, then a homozygous (RR) and heterozygous (Rr) plant can now be distinguished without having to go through progeny testing. Similarly, a homozygous (RR) and a hemizygous (R-) GMO can also be distinguished from one another.

In real time PCR, the results obtained are always related to a control. The copy number of the endogenous gene control should be well characterized and demonstrated in a variety of plant sources. The expected size of the amplified product for an endogenous control obtained must agree with the results expected in the researcher’s experiment in order for the entire experimental data to be considered valid. If results obtained from the endogenous control are not as expected, then results obtained for other target DNA sequences in the same experiment are considered invalid. This critical factor defines the reliability of obtained data in each real time PCR procedure.