What effect does the large temperature difference between the two primers have on PCR, including real time PCR? If I did not get a result from real time PCR, is it possible that my problems are due to a large temperature difference? Can you guide me? What can I do to get results?
If one primer anneals at a much higher temperature than the other then to get a pcr to work the annealing temperature of the lower annealing must be used. This means that the higher annealing primer is being used greatly below its specific annealing temperature so will probably bind non specifically at many places in the genome that fit to part of its sequence so you will get a lot of non specific bands. Check you amplifications on ordinary pcr and see that you get a clean amplification. While you are doing that design a primer pair that have similar annealing temperatures . Primers are cheap.Your time is not.
when you are designing primers try to make it to closer annealing temperature of both forward and reverse primer. I mostly preferred with around 60oC. better to designing new primers with carry out your PCR. ordering of new primers must be cheaper than trial an error with qPCR chemical. Good luck
The principle of work of the pcr and real time is depending on the temperature, in which a difference from the optimum temperature will not get adequate results .. So you have to adjust the temperature according to the method of work.
Smaller difference in melting temperature of two primers eases the annealing temperature choice and increases specificity of the primers, for larger difference in melting temperature, you mainly consider the primer of lower melting temperature when computing for anealing temperature.
Basically to set up the annealing temperature between forward and Reverse primers depend on the total number of nucleotides of each primer {(G+C)x4) + {A+T)2)}. the difference between them is about degree.
High temperature (75- 85) degrees is required to break hydrogen bonds for possible addition of primer. After which the temperature is reduced to about 65degrees for reannealing of the primer and DNA strand. Polymerase enzyme is temperature sensitive and high temperature could reduce copy number.
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