The primer must be between 18 to 22 nucleotides long, having 50% to 55% GC content and can not form the primer dimers. 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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