How TaqMan Works -- Ask TaqMan® Ep. 13

Описание: Submit your real-time PCR questions https://www.thermofisher.com/us/en/ho...
Just how does TaqMan work? Sr. Field Applications Specialist Doug Rains explores the specific mechanism by which TaqMan® achieves its unparalleled specificity and sensitivity. Around the world, researchers rely on TaqMan® for gene expression, SNP gentoyping , protein expression, pathogen detection and quantification, and more.
For many years, TaqMan has been the gold-standard chemistry for real-time PCR. It's famed for its unparalleled specificity, sensitivity, and ease of use. So it's not surprising that users want to know what Shrikant at ICL College in India asked recently: namely, "How does TaqMan work?" I'm glad you asked.
Just like any PCR, TaqMan-based reactions require a double-stranded template, as well as two fairly standard, target-specific primers. But unlike those used in regular PCR, TaqMan Assays require a third, sequence-specific oligo called a probe. TaqMan probes are quite different from the primers in two ways. First, they can't be extended by our friendly enzyme, Taq Polymerase, since they lack a free hydroxyl group.
What's more, TaqMan probes are covalently joined to two other molecules. On the 5'-end, there's a fluorescent molecule known as the reporter -- called that because it reports signal to us as we generate more and more product. On the 3'-end is a molecule known as the quencher, which quenches the fluorescent signal from the reporter under certain circumstances. Let's see what those circumstances are.
Here we're looking at an intact probe, with the reporter in green, the quencher in red. Normally, when we zap the probe with light, we expect the reporter to get excited and fluoresce. But because the quencher is in close proximity to the reporter, instead what happens is, the energy gets transferred from reporter to quencher. The transfer of energy is known as FRET, or Fluorescent Resonance Energy Transfer. The important thing to note here is that, as long as the probe remains intact, there is no permanent increase in fluorescent signal from the reporter.
However, if the reporter and quencher are permanently separated during the reaction, and then light strikes the reaction, the Reporter does in fact fluoresce, producing signal the instrument can detect.
The basic idea, then, is this: each time we create a new PCR amplicon, we want to permanently split the reporter and quencher. By doing so, florescence will always increase proportionally with product, allowing us to effectively monitor what's happening to our reactions throughout the run. Here it is in action.
We begin our reactions (CLICK) by denaturing our template at a high temperature. As we lower the temperature, our probe and primers bind. Taq now comes in, finds the primers, and begins the extension phase of PCR by creating new complementary strands of DNA. But wait a second: there's a probe sitting in the way. It's a showdown in the making! What will the polymerase do? Stop in its tracks? Turn back in fear? Nay, friends, not Taq Polymerase. You see, our enzyme has what's referred to as "exonuclease activity." Meaning? It pretty much eats DNA for lunch.
So when Taq reaches the probe, it simply chews it to bits on its way to creating new amplicon. As a result, the reporter and quencher are physically separated, creating a permanent increase in fluorescence that, not coincidentally, perfectly accords with our doubling of product.
And, of course, that our real-time instrument can monitor and record this increase in fluorescence after each cycle, generating an amplification plot that's more than a little useful for interpreting our data.