It is easy to design Cliffhanger primers as new primers or to redesign already existing primers. Cliffhanger primers are always designed as ZNₓ₁ZNₓ₂ - where the Z designates TINA, Nₓ₁ is the capture sequence of the primer and Nₓ₂ is the complementary part of the primer to the target. The 5' TINA protects the primer from degradation in DNA samples from complex sources e.g. feces and soil.
The internal placed TINA has two functions 1) to increase the annealing temperature of the primer and reduce the primer concentration up to 50% compared to conventional DNA PCR primers - without negative effect on PCR sensitivity. 2) to block the DNA polymerase, thus leaving the capture sequence as a single stranded overhang on all PCR amplicons - ready for direct detection on a solid-phase platform e.g. microarray or Luminex® instrument.
These features enable a higher degree of design-freedom when designing Cliffhanger-PCR assays. Furthermore, Cliffhanger-PCR can be multiplexed with "no upper limit of number of targets" as microarray can contain thousands of capture sequences - or between 1 and 500, if using a Luminex® instrument (model dependent).
The Nₓ₁ is the custom designed capture sequence used for the direct detection on a solid-phase platform. We recommend a length of approximately 20 nucleotides, with no complementarities to DNA sequences found in the sample material.
The Nₓ₂ sequence is complementary to the target of interest and follows the normal rules of designing a PCR primer described under the home page tabs "Design of TINA primers", "End-point PCR" and “Multiplex PCR".
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.
Please always redissolve lyophilized Cliffhanger primers overnight in the fridge before use to ensure that the more hydrophobic TINA modified primers are fully in dilution.