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Phases of RealTime PCR and Why Theyre Important – Ask TaqMan® Ep 9
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Submit your Real-Time PCR questions and watch the rest of our videos at http://ow.ly/bQh0l. Life Technologies Sr. Field Application Specialist Doug Rains helps with the understanding of the different phases Real-Time PCR and why they're important. PCR does in fact have phases. But it's normally difficult to see PCR's phases in action. That said, once we do real-time PCR, we can easily visualize its phases. Fortunately, real-time is where PCR phases really matter. When we first start our reactions, there's a molar excess of all the reagents needed for amplification. As a result, we typically get a nice doubling of product with each cycle. In an amplification plot set to log scale, we see that exponential increase in product manifests itself as a straight line. This phase is known variously as the geometric, log, or exponential phase. Unfortunately, this doubling doesn't go on forever. At some point, the reagents in the tube— primers, dNTPs, and so on— start to run low. As a result, the reaction slows down. We call this the linear phase. • Finally, after some point, assuming the plate is run for enough cycles, the reactions run out of some critical ingredient. As a result, the reactions come to a halt. We call this the plateau phase. So why do we care? Because only one phase yields high-quality quantitative data: namely? You guessed it— the geometric phase. To see why, let's look at an example file. • In this run, I took a highly concentrated sample and serially diluted in 10-fold increments over 7 logs. I also ran eight technical replicates for each dilution point. What I want you to notice is this: in the geometric phase, which is approximately the area inside the box, all of my dilutions are very evenly spaced, exactly as I would expect. Also, my pipetting replicates are very tight, meaning I have excellent precision. But as we move into the later cycles—the so-called linear phase— that predictability starts to go away. My nice, straight lines begin to curve, messing up my even spacing. And my precision drops off considerably. Finally, in the later or plateau cycles, my data all merge together. The point is we always want to look at our data in the geometric phase, since this is where we'll get the best results, especially for a quantitative reaction. How do we do that? By making sure that's where the threshold is set.
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