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TINA in End-point PCR

End-point Polymerase Chain Reaction (PCR) is often used as a "toolbox method" for amplification of waste amounts of starting material, requiring no specific optimization. But in some cases a sensitive end-point PCR amplification is needed and such assay will benefit from the use of TINA modified primers.

In end-point PCR, the TINA modification is always placed at the 5' position of the primer sequence and increases the melting point (Tm) of the primer by 3 to 4°C in average. So far all tested end-point TINA modified PCR primers have been found to increase Tm allowing PCR amplification at stressed PCR conditions compared to unmodified end-point PCR primers.
 
TINA MODIFIED END-POINT PCR PRIMERS OFFERS

  • Improved assay multiplexing capacity. TINA modified primers reduce the necessary primer concentration by 30-50% and increase the optimal annealing temperature by 3 to 4 °C in average. This reduces complexity of a multiplex assay and improves assay specifications(1)
  • Improved assay robustness. TINA modified primers improve PCR robustness as "uncleaned" DNA preparations as well as higher background of genomic DNA is tolerated by TINA modified primers compared to unmodified primers(1)
  • Flexibility in assay design. As TINA modified primers increases the Tm of the primer, shorter TINA modified primers perform equally to longer unmodified primers. This is of special interest if variation in the target sequences limits the placement of end-point PCR primers. The TINA modification requires no special primer design and no need for re-design of existing functional primers

 
YOUR BENEFITS
If you are designing multiplex end-point PCR assays, working with restriction in primer design due to target sequences or working with clinical samples, you will be able to take advantage of the TINA technology, as you can significantly reduce the amount of primers in the reaction and you can test less purified sample DNA. This will result in improved PCR efficacy (increase robustness, shorter PCR times and stable performance over a wide range of primer concentrations and annealing temperatures). If you are working with highly multiplexed end-point PCR, then please refer to TINA in multiplex PCR (and our proprietary Cliffhanger® Technology.

 

 
Figure Legend: Amplification of an octaplex end-point PCR by unmodified primers and 5' TINA modified primers. The red box highlights the lack of amplicons for the estAh gene with unmodified primers. 10-fold target dilution series for an Escherichia coli strain entailing three target genes(1).
 
 

(1) Schneider UV, Mikkelsen ND, Lindqvist A, Okkels LM, Jøhnk N, Lisby G (2012) Improved Efficiency and Robustness in qPCR and Multiplex End-Point PCR by Twisted Intercalating Nucleic Acid Modified Primers. PLoS ONE, 7(6): e38451 (gerne som hyperlink til artiklen under news publications).