a cry for help, work style
okay, seriously, we've been working on this problem, and we're stuck.
If you're science-minded at all, please read on. If you're good at PCR, or know anything about it, or you know anything about enzyme activity, I beg you to read on. Look, I'm groveling, OK? If you don't fall into any of these categories, feel free, but it might be a little technical.
Without going into too much detail (because it's industry, so it's proprietary, so i can't), here's the scenario. when we do PCR on this one target, it holds at 72°C as a post-cycle. and we get really lousy results with our detection of the PCR product. (If it matters, detection involves denaturation of PCR product in NaOH and an ELISA-style detection. It's complicated, but it's pretty accurate.) During an experiment, we changed the post-cycle to 90°C. And now, suddenly, we've managed to double the amount of product.
But wait, it gets weirder.
We ran it through a gel analyzer, and there is approximately double the amount of product with the 90°C hold than there is at the 72°C. My first response was that, well, DNA denatures at around 90°C, but the probe used in the analysis was specific to double-stranded DNA, so at least according to that analysis, there is actually double the product, and not just denatured DNA.
However, today we did an experiment where we added an extra cycle and held it at 72°C, but it didn't come anywhere near the results with the 90°C hold. So it's not simply going through another cycle. It also appears to happen in the 45 seconds that it takes to get from 72°C (also the extension temperature) to 90°C; the results are the same whether it's taken out of the cycler immediately or allowed to sit for 15 minutes (the maximum time at the final hold).
There are two enzymes involved in the assay. rTth (because it's an RNA assay) is the polymerase. UNG (Uracil-N-glycosylase) prevents contamination prior to the assay (because our PCR mix uses dUTP instead of dTTP), but it doesn't completely inactivate or denature, so we do know that it does sometimes activate between cycles in certain products; however, it's supposedly not active above 55°C.
That's as far as we've gotten so far. Anything you can come up with, even if it's a shot in the dark, will be accepted. We have no clue what's going on, so we'll try anything once. We've even thought about nanobots taking nanopictures, as though it were possible. :)
Please comment to me with any ideas you might have.
Feel free to point other people you know to this entry. The more ideas I can get, the better off I am. I was vague enough that it can be shared as much as needed.
Thank you all for your help!
If you're science-minded at all, please read on. If you're good at PCR, or know anything about it, or you know anything about enzyme activity, I beg you to read on. Look, I'm groveling, OK? If you don't fall into any of these categories, feel free, but it might be a little technical.
Without going into too much detail (because it's industry, so it's proprietary, so i can't), here's the scenario. when we do PCR on this one target, it holds at 72°C as a post-cycle. and we get really lousy results with our detection of the PCR product. (If it matters, detection involves denaturation of PCR product in NaOH and an ELISA-style detection. It's complicated, but it's pretty accurate.) During an experiment, we changed the post-cycle to 90°C. And now, suddenly, we've managed to double the amount of product.
But wait, it gets weirder.
We ran it through a gel analyzer, and there is approximately double the amount of product with the 90°C hold than there is at the 72°C. My first response was that, well, DNA denatures at around 90°C, but the probe used in the analysis was specific to double-stranded DNA, so at least according to that analysis, there is actually double the product, and not just denatured DNA.
However, today we did an experiment where we added an extra cycle and held it at 72°C, but it didn't come anywhere near the results with the 90°C hold. So it's not simply going through another cycle. It also appears to happen in the 45 seconds that it takes to get from 72°C (also the extension temperature) to 90°C; the results are the same whether it's taken out of the cycler immediately or allowed to sit for 15 minutes (the maximum time at the final hold).
There are two enzymes involved in the assay. rTth (because it's an RNA assay) is the polymerase. UNG (Uracil-N-glycosylase) prevents contamination prior to the assay (because our PCR mix uses dUTP instead of dTTP), but it doesn't completely inactivate or denature, so we do know that it does sometimes activate between cycles in certain products; however, it's supposedly not active above 55°C.
That's as far as we've gotten so far. Anything you can come up with, even if it's a shot in the dark, will be accepted. We have no clue what's going on, so we'll try anything once. We've even thought about nanobots taking nanopictures, as though it were possible. :)
Please comment to me with any ideas you might have.
Feel free to point other people you know to this entry. The more ideas I can get, the better off I am. I was vague enough that it can be shared as much as needed.
Thank you all for your help!