Suppose the annealing temperature for our PCR is 55☌. Now, let us understand the process by taking one simple example.
In the touchdown PCR, the annealing temperature is selected 10☌ higher than the melting temperature. To overcome this problem, the touchdown PCR is designed. It is hard to calculate the exact annealing temperature for impossible templates such as high GC-rich DNA.Īll these problems result in non-specific unwanted amplification. Hence it might possible that at a given annealing temperature the primer may bind to the sequence other than its complementary sequences. Generally, the annealing temperature is 5☌ lower than the melting temperature but it is just an approximate assumption. So ideally, the annealing temperature should be lower than the melting temperature, and then the primers can bind to its complementary sequence. Whereas, the melting temperature is the temperature at which the primer dissociates from the complementary DNA sequence.Īlso, the melting temperature is defined as the temperature at which half of the DNA strand is dissociated or denatured. The annealing temperatureof the primers is the temperature at which the primers bind to their specific complementary sequence on DNA. The annealing temperature of the primers must be between 55☌ to 65☌ (ideally). The primer must be between 18 to 22 nucleotides long, having 50% to 55% GC content and can not form the primer dimers.
DESIGNING TOUCHDOWN PCR FREE
The primers are the set of the short single-stranded DNA used in the PCR reaction which facilitates the amplification by providing the free 3’-OH ends to Taq DNA polymerase.įor more detail on the DNA primers, read the article: PCR primer design guidelinesįor that, we have to design our target sequence-specific primers by using online tools. The touchdown PCR is the modification of the conventional PCR in which the high specificity of amplification is achieved by reducing the unwanted amplification on sequentially decreasing the annealing temperature after each PCR cycle.įor understanding the basic concept of the touchdown PCR, we have to understand the mechanism of how the primers anneal to the PCR template DNA. In this article, we will discuss touchdown PCR which facilitates high specificity in any PCR reaction. Is there any technique that avoids unwanted amplification and is still a success in the PCR reaction?
Increasing the annealing temperature failure in PCR reaction can also be possible. The graphical representation of specific and non-specific bindings at the higher and lower annealing temperature, respectively.īy increasing the annealing temperature, the primers cannot bind to other than its complementary sequence and result in specific amplification, however, it is not always the case. Hence it is necessary to stop any unwanted amplification in PCR reaction. Non-Specific bindings are the amplicon other than the target sequence amplification. Primer-dimers and non-specific binding are two major setbacks of any PCR reaction or we can say it is a kind of curse for any PCR reaction. The unwanted amplification, a non-specific binding results in false-positive or false-negative results.Īlso, the unwanted primer-dimer amplification results in the shorter non-specific bands which makes the primers unavailable for the target amplification. In the touchdown PCR, “By sequentially decreasing the annealing temperature during each PCR cycle, the chance of the non-specific binding can be reduced.”Įvery PCR technique is evolved to eliminate unwanted amplification during the PCR reaction.
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