High Fidelity PCR
The fidelity of polymerase chain reaction (PCR) refers to the accuracy of DNA sequence replication, which depends on factors such as polymerases, reaction conditions, buffer systems, and the quality of the template DNA. The selection of appropriate concentration of magnesium ions (Mg²⁺), annealing temperature, denaturation time and temperature, extension time, cycle number, and primer design are very critical when optimizing reaction conditions to improve the fidelity.
Polymerase-independent Influence Factors
An excessive number of cycles will increase the accumulation of errors. In the later stage, when the template volume is high, the error rate may rise exponentially. The quality and concentration of the template DNA are also very important for the fidelity of PCR. Because the less DNA is at the beginning, more cycles are needed to obtain the same amount of the final product. With each additional amplification cycle, the already existing errors will be copied and consequently doubled. In addition, damaged or degraded DNA template can lead to replication errors. When DNA is exposed to high temperatures, it will be damaged and cytosine will be deaminated into uracil, which leads to the mutation from C-G to T-A. To avoid this situation, choose a short denaturation time and, if possible, completely omit the initial denaturation step.
Polymerase-dependent Influence Factors
To minimize the errors of polymerase dependence as much as possible, conditions that promote slow elongation should be selected. Because the slower the elongation rate of the polymerase is, the more time is to ensure the the incorporation of the correct nucleotides. High-fidelity polymerases with proofreading functions are ideal to remove mismatched bases, significantly reducing the error rate.
AQ97 High Fidelity DNA Polymerases
AQ97 high fidelity (shortened to HiFi) DNA polymerase is a novel proofreading DNA polymerase, and is composed of a novel chimeric DNA polymerase with archaeal ancestry, fused to the processivity-enhancing DNA binding domain. In addition to very fast and robust amplification of complex and long targets including those up to 18 kb and with high to low GC content, the AQ97 HiFi DNA polymerase can ensure accurate amplification. AQ97 HiFi DNA polymerase is well suited for PCR experiments that require amplification with very low error rates, such as cloning/subcloning, NGS applications, SNP analysis, and mutagenesis. The fidelity of the polymerase has been measured to be more than 60x Taq DNA Polymerase.
Product Range
Amerigo Scientific offers two variants of AQ97 HiFi DNA polymerase, including AQ97 HiFi DNA polymerase and AQ97 hot start HiFi DNA polymerase. Both products are available in a 2x master mix version. The hot-start version of AQ97 HiFi DNA polymerase can be set up at room temperature without the risk of primer degradation and reduced specificity. In addition, we also provide AQ97 HiFi hot start 2x master mix with red dye, increasing the convenience for the user even further.
Amerigo Scientific also provides AccuPOL DNA polymerase that is a thermostable high-fidelity DNA polymerase with proofreading capability. AccuPOL DNA polymerase without buffer and AccuPOL DNA Polymerase, 10x Ammonium Buffer and MgCl2 are available. The AccuPOL DNA polymerase is recommended for cloning, mutagenesis and when blunt ends are required.
Features
All these DNA polymerases are manufactured in-house in accordance with the ISO 9001:2015 quality management system, ensuring the same robust performance per batch every time.
- High fidelity: > 60x Taq fidelity
- High elongation rate: 10 sec/kb
- 3’ to 5’ proofreading exonuclease activity
- Hot start version: reaction setup at room temperature
- Long range amplification: 18 kb for human gDNA and 25 kb for λ DNA
Technical Characteristic
Robust Amplification of AQ97 Hot Start HiFi DNA Polymerase and Master Mix
Performance of AQ97 HiFi Hot Start DNA Polymerase (AQ97 HS HiFi) was compared to a leading hot start high fidelity DNA Polymerase Q. Eight different human genomic DNA targets, 400-800 bp in length and with GC content ranging from 29-78%, were amplified. Robust amplification was observed for all targets using AQ97 HS HiFi. DNA Polymerase Q provided very similar results to AQ97 HS HiFi. When amplifying GC-rich targets, 2 M Betaine Enhancer Solution (AQ97 HS HiFi) or a GC enhancer (DNA Polymerases Q) was included in the reaction mix.
Figure 1. Comparison of the performance of AQ97 HS HiFi with that of the hot-start high-fidelity DNA polymerase Q
The above comparison was also performed between AQ97 HiFi Hot Start 2x Master Mix and the leading Hot Start High Fidelity 2x Master Mix Q. Robust amplification was observed for all targets using AQ97 HiFi Hot Start 2x Master Mix and Hot Start High Fidelity 2x Master Mix Q. For the target with 78% GC content, a robust amplification with high yield using AQ97 HiFi Hot Start 2x Master Mix was observed, whereas this target could not be amplified using Hot Start High Fidelity 2x Master Mix Q.
Figure 2. Comparison of the performance of AQ97 HiFi Hot Start 2x Master Mix with that of Hot Start High Fidelity 2x Master Mix Q
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