ThermaGenix PCR Additives Reduce Background and Increase Yield

Easy-to-Use PCR Additives

Reducing non-specific background and increasing PCR yield

ThermaStop™, ThermaGo™ and ThermaStop™-RT reagents are easy-to-use PCR additives that act at different temperatures (Figure 1). Unlike hot start PCR, ThermaStop™ and ThermaGo™ additives suppress errors before, during, and after amplification for reactions starting with either DNA or RNA, therefore enhancing PCR specificity and sensitivity. These PCR additives make multiplexed diagnostic reactions more accurate and informative, and also enhance the quality of all downstream applications, including next generation sequencing (NGS), deep sequencing, and Gibson assembly. The ThermaStop™-RT additive is also a double-stranded nucleic acid, however it acts as a hot start for reverse transcriptase during one-step RT-PCR and two-step RT-PCR.

Figure 1. PCR Additives used at Different Temperatures

* ThermaStop™-RT should not be used in combination with ThermaStop™ or ThermaGo™ additives

Risks of mispriming at different steps and temperatures during amplification

Preventing mis-priming improves PCR amplification results. Refining the success of PCR reactions depends on specificity and efficiency. The ThermaStop™ reagent acts before and after amplification while ThermaGo™ reagent is active during the denaturation, annealing and extension steps of PCR (Figure 2).

Figure 2. PCR Additives Used at Different Steps to Prevent Mis-Priming during PCR

The ThermaStop™ additive is an innovative hot start and cold stop reagent to enhance Taq DNA polymerase specificity and to improve PCR product yield

The ThermaStop™ PCR reagent is a reversible additive compatible with all Taq DNA polymerases, including hot start polymerases. It acts directly on the polymerase to prevent non-specific enzymatic activity below 50 °C. Polymerase activity is fully restored by 60 °C, but is inhibited again by cooling the reaction.

• Enables master mix long-term storage
• Significantly reduces primer-dimer formation
• Facilitates PCR multiplexing
• Increases reaction sensitivity and specificity
• Eliminates undesired products after cool down

Figure 3. The ThermaStop™ additive improves PCR reaction specificity and yield resulting in cleaner amplification.

Figure 4. The ThermaStop™ additive stabilizes master mixes.

Figure 5. The ThermaStop™ additive inhibits DNA polymerase activity following PCR, thereby functioning as a cold stop.

The ThermaGo™ additive enhances Taq DNA polymerase specificity throughout PCR and can act synergistically with the ThermaStop™ additive to increase Taq fidelity

The ThermaGo™ PCR reagent 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.

• Reduces signal scatter in standard and digital PCR
• Increases detection of low-copy number targets by suppressing amplification of non-specific products
• Improves endpoint genotyping

Figure 6. The ThermaGo™ additive is active during amplification, reduces scatter, and increases product signals. Lines represent probe signals from a Taqman probe.

Figure 7. The ThermaGo™ additive enhances the ThermaStop™ additive in multiplexing applications by preventing mis-priming before and during amplification.

The ThermaStop™-RT additive improves RT-PCR sensitivity, specificity, and product yield

ThermaStop™-RT reagent is for one-step or two-step reverse transcription-PCR (RT-PCR) initiated with gene-specific primers.

• Interacts with the reverse transcriptase at low temperatures to reduce priming errors that lead to non-specific products
• Improves detection sensitivity and increases specific cDNA yield
• Enhances accuracy of highly multiplexed RT- PCR assays
• Permits quantitatively accurate measurements of very low levels of RNA

Figure 8. The ThermaStop™-RT additive is active during the reverse transcription step to prevent mis-priming.

Figure 9. The ThermaStop™-RT additive improves real-time detection of cDNA.

The ThermaStop™, ThermaGo™, and ThermaStop™-RT additives are simple and universal reagents that can be added to master mixes to improve your product yield and specificity in PCR amplification. These easy-to-use reagents do not require optimization for use with different enzyme systems, so you can start improving PCR specificity and yield immediately.