AQ97 High Fidelity DNA Polymerase 2 U/µl is a proofreading DNA polymerase displaying the following features; high fidelity >60x Taq DNA Polymerase, fast amplification due to its high elongation rate, ability to amplify problematic DNA targets, such as those with low to high GC content and ability to perform amplification of long DNA targets. These features enables accurate and reliable PCR results.
AQ97 High Fidelity DNA Polymerase is recommended for applications, which require extremely high fidelity such as cloning/Sub-cloning, NGS applications, SNP analysis and mutagenesis.
FEATURES
High fidelity: >60xTaqFidelity
High elongation rate: 10 sec/kb
3’→5’ proofreading exonuclease activity
Long range capacity: 18 kb for gDNA and 25 kb for λ DNA
Good coverage on DNA templates with low to high GC content
DESCRIPTION
AQ97 High Fidelity DNA Polymerase exhibits both 5'→3' DNA polymerase activity and 3'→5' proofreading exonuclease activity enabling this polymerase to correct base pair mismatches. The DNA binding domain of this polymerase, ensures the excellent high fidelity, long range capacity and also fast amplification results.
The AQ97 High Fidelity DNA Polymerase kit is supported with 5x AQ97 Buffer allowing robust amplification on DNA targets with low to high GC content and long DNA targets. The kit also includes an extra tube of MgCl2 in 25 mM concentration if adjustment of PCR conditions is required.
For difficult amplicons, such as GC-rich DNA samples, those with complex secondary structures or longer amplicons, the addition of 1 – 2 M Betaine Enhancer Solution is recommended.
For more convenient handling AQ97 High Fidelity DNA Polymerase is also available as a 2x master mix.
FIDELITY
The fidelity of a polymerase refers to its ability to insert the correct base during PCR. On the contrary, the rate of misincorporation is known as a polymerase error rate. Fidelity depends on the polymerase, the buffer system in use and the quality of your template DNA.
FIDELITY: The fidelity values of AQ97 High Fidelity DNA Polymerase, AccuPol DNA Polymerase, high fidelity DNA polymerase P and high fidelity DNA polymerase Q was compared to the fidelity value of Taq DNA polymerase (1x)
ERROR RATES: a Errors per base per doubling. Standard deviations are given in (). The presented error rates may not be comparable to those presented elsewhere due to technical and methodical differences. b Error rates for AQ97 High fidelity DNA Polymerase and also for the two high-fidelity DNA polymerases P and Q were below the detection limit for the method used. This limit is estimated to be 8.4 x 10-6 errors per base per doubling (60 x Taq fidelity). Click to enlarge picture.
LONG-RANGE AMPLIFICATION
AQ97 High Fidelity DNA Polymerase enables amplification of large and complex amplicons. Six different targets of human genomic DNA ranging from 2 kb and up to 17.5 kb was used in this study. Amplicon sizes are indicated at the top of the gel. Marker M is High Range DNA Ladder from Ampliqon (A610141). Click to enlarge picture.
ROBUST AMPLIFICATION
Performance of AQ97 High Fidelity was compared to three leading high-fidelity DNA Polymerase (Q,S and P). 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 High Fidelity DNA Polymerase. High fidelity DNA polymerase Q and S provides results very similar to AQ97 High Fidelity DNA polymerase, except on the last target with the highest GC content of 78%. In contrary, high fidelity DNA polymerase P were not able to provide the same level of robust amplification on the DNA targets with higher GC content, under the conditions tested here. Amplification studies has been set up, as recommended by the manufactures. Tm calculators of the respective competitors were used to calculate optimal annealing temperatures for primers. When amplifying GC-rich targets, 2 M Betaine Enhancer Solution (AQ97 DNA Polymerase), GC enhancer (Competitor Q and S) or GC-rich specific PCR Buffer (competitor P) were included in the reaction mix. Click to enlarge picture.
LOW NUMBER OF SUBSTITUTIONS
Distribution of substitution errors. PCR was performed using Taq DNA Polymerase, AQ97 High Fidelity DNA Polymerase, high fidelity DNA Polymerase Q and high fidelity DNA Polymerase P. The PCR was followed by NGS sequencing of the PCR products. The number of substitutions at each PCR target position was calculated and plotted in diagram A. Substitutions include misincorporated nucleotides and deletions at each position. Non-polymerase errors are subtracted from the total number of errors to revel true polymerase errors. Non-polymerase errors include mutations caused by thermocycling-induced DNA damage, pre-NGS sample preparation and sequencing errors. In these diagrams the average number of substitutions for Taq DNA Polymerase (Taq average) and for AQ97 High Fidelity DNA Polymerase (AQ97 average) is also plotted. Diagram B magnifies the area near the detection limit, displaying more information about the number of substitutions for AQ97 High Fidelity DNA Polymerase, high fidelity DNA Polymerase Q and high fidelity DNA polymerase P. Click to enlarge diagrams.
