Dear all,
I am currently designing an ARMS-qPCR assay to detect mutant alleles at very low levels while diluted with a high number of wild type alleles in the same solution.
I first did a gradient PCR on a real time PCR thermal cycler between 56 to 61.5 degrees Celsius to first determine the best annealing temperature for my primers. This was determined by the slope of the exponential phase of the amplification plot in log view. I believe a steeper gradient implies greater PCR efficiency, which implies a better annealing temperature. All annealing temperatures allow for a similar plateau height, so I believe the amount of amplicons produced at the different Tms are also similar.
From that gradient PCR experiment, I chose 60C as the optimal annealing temperature. I then diluted the template and ran a qPCR experiment with that Ta, this time to determine the PCR efficiency.
Between each 10 fold serial dilution, the Ct difference was 5. Consequently, the calculated PCR efficiency was poor (57%), but I had a very good R2 value of 0.99.
Since I have already determined the optimal annealing temperature for the primers, is this truly the best PCR efficiency I can achieve with the current primers?
From what I understand, poor efficiency can lead to poor analytical sensitivity (high limit of detection). Is this true as well?
Thank you in advance.
Remember everything in qPCR is log based 2; the amplicon should theoretically double with each cycle (in log-linear phase). 10 = 2^3.32192, therefore the slope of your curve for a reaction with 100% efficiency is -3.32192. Meaning for every 1:10 dilution the Ct should be ~3.2 cycles later. A slope of -5 (irrespective of good correlation) means that with each 1:10 dilution the product is coming up 5 cycles later (2^5 ~ 1:32), which is why your efficiency is so low (efficiency = 10^(-1/slope)-1 * 100). 57% efficiency is too low for a quantitative assay; 85%-110% is generally considered the acceptable range. What are you using as the standard? Generally poor efficiency indicates either an inhibiting factor in the sample (that often resolves itself with dilution and is observable as the curve is flat at the top) or poor assay/primer design. The reality is some amplicons are just difficult to PCR (high GC content ect). Your temperature gradient is rather narrow, have you tried anything broader? 50-65C, getting at the GC issue? Also, are you using a commercial qPCR Master Mix or a homemade recipe? Many commercial kits have "secret" ingredients that help PCR difficult targets (e.g. DMSO, betaine). Also in that vein, if you're using a home made MM have you optimized for Mg2+? Last but not least, are you sure you did a 1:10 dilution? You don't know how many times I've looked at an assay and been dismayed by my efficiency and then realized the dilution I did was different than I thought.... Saved from my own ineptitude with good notes;)
As to your method of determining your annealing temp, better ways to do it, you are making assumptions you shouldn't be. I agree with Laura, expand out the temperature range a little bit and be certain to add a melting curve! This will tell you whether you are forming any products you don't want to be forming. The goal is to use the lowest temperature that you can that doesn't form any product but what you want (too high a temperature and you won't get all of your primers annealing nicely).
You may also need to play with the primer concentrations. It may sound counter-intuitive, but you don't want your concentration of primers too high. Your reaction may form primer-dimers or eat through your dNTPs too quickly.
The height of your plateau is irrelevant. it is a factor of your primer concentration (assuming you have a non-negative sample).
While on the topic of primers, have you run them through any of the algorithms to determine secondary structures and self-annealing tendencies? It's worth doing. It may help you figure out if you need to have different concentrations of your primers or if you need to redesign your primers.
Next, don't be afraid to play with you extension time, especially if you have a sizeable amplicon. Chances are, this won't effect anything, but don't be afraid to use a longer extension time.
Honestly, the best way to analyze your gradient PCR is to run out some of your PCR products on an agarose gel and look at band intensity. No, you have NOT determined the best annealing temperature. Go back and try again.
57% efficiency is terrible. You can not use these data. Publishable efficiencies are between 95-105%. The 99% R^2 means you have good pipetting skills - nice job!
You will probably need to test several sets of primers to find ones with a a useful annealing temperature.
Things to modify once your efficiency is CLOSE to 95-105%
- Primer concentration
- magnesium concentration
- annealing temperature
- template concentration
Good luck!
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