Non-specific amplification is one of the greatest challenges for the successful deployment of real-time PCR methods intended to be used for the validation or discovery of transcript abundance variation. In addition to the sequence of interest, many thousands of other sequences can be expected to be present in such applications. The design of PCR primers for this purpose should therefore take into account the potential contribution of all possible off-target template sequences, in order to prevent mispriming. This is usually achieved by comparing sequence similarity between the primers and all other template sequences in the design space.
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