User talk:570ceh
Hello, good questions. I'll see if I can answer them. I copied them below because I couldn't figure out any other way to see them while I reply!
Hi! I have been doing some work with using real time PCR for detection of Salmonella in environmental samples, so I know just enough to be dangerous. Can you explain to me why working with the amplified PCR product from nested PCR could produce false positives? Nested PCR involves an extra handling step mid-test.....after the first round of amplification is done (potentially already creating thousands / millions of copies of cDNA), those tubes are opened and transferred to another tube where another round of amplification is done on a smaller region within the larger cDNA. Basically, when you have to open / close tubes that likely have tons of amplified DNA, you run the risk of that material getting into places it's not supposed to go. Now, contrast that with real-time PCR in which you add only template DNA or RNA (as in the material you extracted from a clinical sample) and close the tube and let amplification go....then never open the tube again while the results are read through light emitted through the tube. Make sense?
Also, it was my understanding that the Taqman probes used for real-time PCR bind to double stranded DNA which results from other steps in the amplification process, but that the probes were not specific to a certain sequence themselves. Not quite....although their are variations of real-time chemistry BESIDES taq-man which fit what you are saying. But for taq-man probes, they most definitely target a specific sequence within the overall amplicon (fancy term for the part of the DNA you amplified with primers in the PCR). Anyway, taq-man probes bind to the single-stranded DNA during the extension step and are cleaved (torn apart) by taq-polymerase as it makes a new copy of DNA. After being cleaved they can be detected by real-time PCR equipment. Again, there are other forms of real-time chemistry out there....but for PRRS it's currently all done with Taq-man probes. Sybr-green, for example, is non-specific.
I am a geologist trying to learn microbiology without a strong grasp of the fundementals, so forgive me if this is a stupid question! 570ceh (talk) 16:52, 8 November 2011 (UTC) No worries, thanks for the questions! I love talking about this stuff, would be happy to chat sometime. 515-294-6968
Awesome! I'm glad you liked it and was able to add to it. Sorry it took me so long to finally add the section, haha. I was sick most of last week and was in a nightquil coma for most of it :) Christi
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