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TINA in Multiplex PCR

The TINA modification is placed at the 5' position in PCR primers as it blocks the read-through by the Taq DNA polymerase.

This feature of the TINA molecule is used in the Cliffhanger® Technology, which enables superior multiplex PCR assays with solid phase detection. In the Cliffhanger® Technology, the TINA modification is placed at the 5' postion of the primer sequence increasing A) the flexibility in primer design, B) assay robustness and C) multiplexing capacity as amplicon length can be optimized allowing equal PCR efficiency regardless of the number of targets. The TINA modification also acts as a linker between the primer sequence and a single stranded DNA overhang, which is beneficial for solid-phase detection and only needs to be optimized once.

 
Figure Legend: A Cliffhanger primer consist of i) a target specific primer sequence, ii) a TINA molecule and iii) a custom designed overhang sequence or label. Each strand in the PCR amplicon contains a single stranded cliffhanger overhang, which can be designed freely.
 

THE CLIFFHANGER® TECHNOLOGY OFFERS:

  • Faster assay development. The custom designed capture oligonucleotide sequences can be reused in all future assays - allowing one single detection panel to cover all future assays and no need for extensive optimization of the solid phase capture for each future assay
  • Simplified assay design. Primer design only depends on target sequence, not subsequent capture and detection oligonucleotides as the single stranded DNA overhangs are custom designed and independent of target sequence
  • Improved assay robustness and efficiency. a) The TINA molecule improves the efficiency and robustness of multiplex PCR assays and b) Cliffhanger PCR primers allow similar amplicon lengths for all targets (uniform multiplex PCR efficiency)
  • Increased assay sensitivity and specificity. a) No need to denature PCR amplicons to allow capture, as specific single stranded overhangs are added to the PCR amplicons by the Cliffhanger primers (no need for formamide or other solvents for denaturation and increased sensitivity as overhangs are single stranded without competing sequences in the reaction) and b) increased assay specificity due to reduced potential for cross-reaction as the cliffhanger overhangs are custom designed and reduced PCR primer concentrations are needed
  • Dual benefits. The Cliffhanger® Technology is the only technology that in one step improves both the multiplex PCR and the subsequent detection of the PCR amplicons

YOU ONLY NEED TO:

  • Establish a single capture panel to get a robust detection array for all future assays
  • Optimize your multiplex TINA PCR primers for each new assay
  • Have a robust nucleic acid extraction and purification step upfront, but your multiplex Cliffhanger PCR will tolerate inhibitors more efficiently than current linker based technology