To ease the design of multiplex PCR assays, it is beneficial if the primer concentrations can be reduced without decreasing the PCR efficiency in order to diminish the likelihood of primer cross-reactivity and amplification of non-target nucleotide sequences.
Likewise, the ability to increase the annealing temperature or decrease the primer length without negative impact on the PCR efficiency is desirable in order to diminish cross-reactivity of the PCR primers and subsequent problems with assay specificity. Additionally, shortening the primer lengths not only increases assay specificity, but also eases primer design for clinical relevant targets by expanding the range of possible target regions. These benefits are especially important for targets with high mutation rates and when designing multiplexed PCR assays.
TINA-PCR primers enable design of multiplex PCR assays with substantially less amount of primers – often, approximately 50% reduction compared to conventional PCR primers can be achieved. Thus, assay complexity is greatly reduced. This can be translated to better efficacy – or increased level of multiplexing - compared to a conventional multiplex PCR assay.
Your initial consideration will be the level of multiplexing. If you choose multiplex real-time PCR, you will be limited by the number of individual fluorescence channels on your chosen real-time PCR platform. Currently, the upper limit is six channels – so your maximum level of multiplexing in real-time PCR will be five targets plus control. Alternatively, you may choose to design a multiplex end-point PCR assay with subsequent detection on a solid-phase platform – e.g. array or Luminex. In this case, there is no fixed upper limit for your level of multiplexing.
When you design primers for multiplex PCR, the goal is to achieve identical optimal Ta for all primers. This must be tested in your assay during the initial optimization phase.
When optimizing a multiplex TINA-PCR, please observe the protocols listed under "End-point PCR with TINA-primers" or "Real-time PCR with TINA-primers". If your initial analytical sensitivity study shows one or more primer set with reduced efficacy, try to adjust primer concentration of these primer sets.