With the availability of more advanced genomic technologies, there’s a directly related need for more sophisticated DNA polymerases to deliver the processivity, sensitivity, and fidelity required for optimal amplification.
The Quantabio team prepared a new application note to describe best practices for PCR amplification using our repliQa HiFi ToughMix, which has been described by academic scientists as the “enzyme of choice” for NGS library amplification after outperforming other products in a benchmarking exercise.
For a top-notch DNA polymerase, key traits include specificity, thermostability, fidelity, processivity, long-range capability, inhibitor tolerance, and ability to amplify high-GC targets. Unfortunately, most polymerases perform well for only a few of these traits. We developed repliQa HiFi ToughMix to address all of these traits — and more.
This product is a unique next-generation PCR master mix that provides 90 times higher fidelity compared to wildtype Taq, while showing extreme speed, with extension times as fast as 1–10 sec/kb depending on target length. It can amplify fragments of genomic DNA as long as 24 kb and with varying GC content. Additionally, the enzyme is coupled with our industry-leading ToughMix technology, making it tolerant to a wide range of PCR inhibitors and able to work with crude lysate or dirty samples without the need for purification steps. Finally, repliQa HiFi ToughMix can amplify from templates containing uracils (dU) or using primers containing inosines (dI) and uracils, making it ideal for applications like DNA methylation analysis or bisulfite genomic sequencing.
We encourage you to check out the application note to learn more about how to use repliQa HiFi ToughMix for a range of PCR amplification needs. Highlights include:
Guidance on cycling conditions: The annealing temperature, extension time, and GC content are critical parameters to consider when selecting the cycling program and conditions to guarantee a successful PCR.
Long-range amplification capabilities: This versatile amplification reagent can be used for both smaller targets and long-range amplification without compromising PCR yield. We tested targets as small as 250 bp and as large as 20 kb.
High and low GC content: We evaluated DNA with GC content ranging from 27% to 67%, amplifying targets of three different sizes for each. repliQa HiFi ToughMix was able to successfully amplify all nine targets.
Whole genome libraries: We put this enzyme to the test, using as little as 10 ng of mixed bacterial genomic DNA with a 1 second extension time. The output was comparable to that from using 100 ng with a standard 30 second extension time amplified with a different master mix, with excellent genome coverage across a range of GC levels.
Overall, repliQa HiFi ToughMix outperforms other high-fidelity polymerases due to its remarkable array of features: inhibitor tolerance, long-range amplification abilities, suitability for wide GC content ranges, and speed.
2min
Apr 25, 2025
