ThermaGo™ PCR Additive

Enhanced Taq DNA Polymerase Specificity Throughout PCR

The ThermaGo™ additive is a double-stranded, chemically modified nucleic acid that suppresses mis-priming during the annealing and extension steps of PCR. It increases the reproducibility of both real-time and end-point analyses. The ThermaGo™ additive enhances the specificity of Taq DNA polymerase.

The ThermaGo™ Additive Suppresses Mis-Priming During PCR:

  • Improves the reproducibility of technical and biological replicates
  • Increases detection of low-copy number targets by suppressing non-specific products
  • Reduces signal scatter in standard and digital PCR
  • Reduces mis-priming and chimera production prior to next-generation sequencing.
  • Improves end-point genotyping

Applications of Interest:

  • Mispriming protection in all forms during PCR
  • Detection of low copy target numbers (single-cell PCR, digital PCR, rare target detection in liquid biopsies/tumor samples)
  • Enhancing accuracy of highly multiplexed-PCR for targeted next-generation sequencing and other post-PCR applications
  • Monoplex and multiplex endpoint SNP genotyping

ThermaGo™ additive reduces scatter, increases product signals, and allows greater separation of signals

Figure 1 - The ThermaGo™ additive reduces scatter, increases product signals, and allows greater separation of signals from homozygous and heterozygous genomes during real-time or end-point analyses. Lines represent probe signals from a Taqman probe against allele A.

Red lines, homozygous AA target
Blue lines, heterozygote Aa target
Green lines, homozygous aa target


How to Prepare for Use:

The ThermaGo™ additive is shipped as a dry reagent in 500 or 2500 units. To prepare 5 Units/μL of the ThermaGo™ additive, add 100 μL molecular-grade 10 mM Tris-Cl, pH 8.3 to 500 units dry reagent (500 μL 10 mM Tris-Cl, pH 8.3 to 2500 units dry reagent), vortex 1-2 minutes, then centrifuge briefly. Allow tube to sit at room temperature for 15 minutes with occasional mixing to ensure reagent is completely dissolved.


Recommended Storage:

Store the ThermaGo™ additive at 4 °C or -20 °C in the dark or in light protected tubes. If frozen, divide stock into small volume aliquots to avoid freezing and thawing more than 5 times.


How To Use:

One unit of the ThermaGo™ additive is defined as the amount required for maximum improvements in specificity, yield, and reproducibility in amplification reactions containing 1 unit of Taq DNA polymerase in a volume of 25 μL. To use, begin by adding an equal number of units of the ThermaGo™ additive and Taq DNA polymerase to a PCR master mix. The ThermaGo™ additive works synergistically with the ThermaStop™ additive to provide improved PCR performance before, during, and after amplification.


Typical 25 μL PCR

Final Concentration Volume Reagent
10X PCR Buffer 1X 2.5 μL
2 μM primers 0.2 μM 2.5 μL
5 U/μL Taq 0.05 U/μL 0.25 μL
5 U/μL ThermaGo 0.05 U/μL 0.25 μL
Template   x μL
H2O   QS 25 μL

To further optimize, test the ThermaGo™ additive in the range of 0.5 to ≥2.0 Units per Unit of Taq DNA polymerase. Note: very high levels of the ThermaGo™ additive can inhibit amplification; thereby reducing product yield.


Expected Results:

Reactions containing optimal levels of the ThermaGo™ additive will exhibit greater quantitative reproducibility due to suppression of non-specific products. Use of the ThermaGo™ additive will produce higher yields of the intended products (e.g. stronger probe signals, correct bands on gels).

Materials

     
Related Links