For example, the parameters for PCR are denaturing at 95, annealing at 58 and elongation at 72, while the melting temperatures of primers are around 60.
That is an interesting question. I don't exactly know the percentage, but I think if you add tons of DNA template, but an extreme low amount of primers in a reaction, then in theory this will cause a low percentage of original templates got amplified (synthesized). For example, you have 10,000 copies of templates, and 10 copies of primers. Therefore, the percentage can be vary and determinated by the amounts of templates and primers you add. DNA polymerase used might also play a role.
That's a good question. I never think about it even though I use PCR for many years. Theoretically speaking, if there is enough primers and substrates, all the template should be used. That's why we see the linear range for the qPCR for the early cycles. anyway, the difference between original plate and synthesized fragment is the latter DNA is not modified with methy.
I think you are actually asking about the accuracy of your PCR reaction.
So, in terms of accuracy, to the best of my knowledge, if you add your PCR components in an ideal amount and give an ideal thermal profile for carrying out the PCR reaction, then you can expect to have your desired PCR product at 100% accuracy.
That is why, we generally consider the PCR fragments are generated in a 2^n times. But the actual formula is-
N = (1+Xe)^n
Here, N = Final no. of fragment; n = cycle number, Xe = accuracy of your PCR reaction.
So, by default, we consider the accuracy as 100% i.e. Xe = 1
For that the formula is changed to N = 2^n
Now this accuracy depends upon some key factors like-
1. Amount of your PCR components
2. Accuracy of your Polymerase enzyme (Taq/Pfu etc.)
3. Annealing temperature (it controls the stringency of your PCR reaction)
4. Time given for Extension (it should be optimum)
Hope this will be helpful.
Thanks,
I think we are talking about PCR efficiency here, not accuracy. If you look for PCR efficiency, you will find plenty of literature on it. Like the one in the link.
Ideally, you have a fully efficient PCR with doubling of your target sequence every cycle. Since you have an exponential phase for quite some cycles in most PCR settings, you will basically amplify all your template until you get out of the exponential phase.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC219490/
I don't know if there is an experimentally proven answer to your question. In theory, if denaturation is complete and there are excess primers and these anneal correctly to the templates, every single one of the target fragments will be amplified for many cycles...that is until one of the reagents becomes exhausted. However, it is possible that some reanealing of the target molecules (I guess that would be a very small number) will occur and this would prevent amplification, even, perhaps at the first cycle: that clearly isn't a major problem as PCR is effective. However, the target annealing problem would increase as the amount of product increases and the relative amount of primers decreases. But why do you want to know???
Yes. You are right. It was my mistake. It should nave been the efficiency instead of accuracy. Thanks for correcting me.
@Yunfu,
You also need to consider the characteristics of your DNA template: for example, with a very high GC content. In that case, even you have trillion copies of primers, it just won't do a thing without adding some PCR helpers (such as PCR additives). This is one of the reasons that no PCR product get amplified at all even you have plenty of templates and excessive primers.
Respected Sir
I don't know exactly But as per my knowledge depending up on number of cycles we run the sample for getting synthesized DNA. However i don't knew exactly weather your DNA sample is genomic DNA or Plasmid DNA.
Thank you
Thank all of you for your comments. I agree with most of yours on the efficiency issue. In my understanding, the exponential production of PCR products is correlated ONLY with the RELATIVE efficiency of every cycle, i.e the same percentage of molecules amplified each cycle, and that has nothing to do with the absolute efficiency. No matter whether the amplification efficiency is 1, 10 or 100% each cycle, you still get the same exponential production assume the efficiency is the same in each cycle (that's probably true). My real question is whether PCR is 1, 10 or 100% in each cycle. Since I am doing linear synthesis or one cycle with one primer, the efficiency close to 100% is critical for my project.
So actually you are not actually doing PCR!!! PCR means chain reaction, indicating an exponential increase in the yield of the product. What you are doing is either a primer-extension (if only a single cycle) or PLR: polymerase linear reaction, so for each cycle you add only one more product. Ten cycles of PCR gives you approx. 1000 times more product, ten cycles of PLR gives you only ten products. You are unlikely ever (well, up to perhaps a million cycles) to run out of triphosphates or primer if you use similar amounts as in a PCR, So the critical question is how much product do you need (and what are you going to do with it!)?
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