A chimeric form of Phi29 DNA Polymerase
4BB™ QualiPhi® DNA Polymerase is a novel, highly processive chimeric form of Phi29 DNA polymerase, expertly engineered for enhanced sensitivity and efficiency.
Phi29 DNA polymerase has been proven to be an extremely processive DNA polymerase (up to more than 70 kb per binding event), with extraordinary strand displacement capacity, coupled with 3’ → 5’ proofreading exonuclease activity, which results in an exceptionally high synthesis fidelity. These distinctive features have been enhanced to produce a DNA polymerase that produces dramatically higher volumes of amplified DNA. This makes our enhanced version of Phi29 DNA polymerase the perfect choice for isothermal DNA amplification, or for any application that requires a high fidelity DNA polymerase with strong strand displacement capacity.
Applications
- Highly accurate DNA synthesis
- Rolling Circle Amplification (RCA)
- Whole Genome Amplification (WGA)
- Amplification of DNA from filter paper blood spot samples
- Protein-primed DNA amplification
- RNA-primed DNA amplification
- In situ genotyping with padlock probes
- Recombination based-cloning
- Proximity ligation assay
Benchmarking
Figure 1: 4BB™ QualiPhi® DNA Polymerase benchmarking against market leading competitors in Whole Genome Amplification (WGA) *600% increase on average yields compared with competitors observed in WGA.
4BB™ QualiPhi® was benchmarked against two of the leading market competitors to compare the resulting DNA yield between the three products. Each product was prepared with varying template volumes in whole genome amplification reactions.
Figure 2: 4BB™ QualiPhi® benchmarking against market leading competitors in rolling circle amplification
The two figures above show the results of comparative WGAs (Figure 1.) and RCAs (Figure 2.) between 4BB™ QualiPhi® DNA Polymerase and two leading market contenders. As is evident in both DNA amplification methods, QualiPhi® Polymerase produces markedly more DNA irrespective of the initial template DNA concentration compared with either of the two leading market competitors.
Quality Control
Absence of endonucleases and exonucleases:
4BB™ QualiPhi® DNA Polymerase has been determined to be free of detectable endonucleases, exonucleases and nicking activity. A fluorogenic substrate designed to react with all these kinds of nucleases has been incubated in the presence of 1μg of polymerase enzyme for one hour at 37°C. No increase in fluorescence compared with the negative control was detected.
Absence of ribonucleases:
4BB™ QualiPhi® DNA Polymerase has been determined to be free of detectable single strand ribonuclease activity. A fluorogenic substrate designed to react with these kinds of nucleases has been incubated in the presence of 1μg of the enzyme for 30 min at 37°C. No increase in fluorescence compared with the negative control was detected.
Purity:
The purity of the enzyme has been determined to be higher than 95% by SDS polyacrylamide gel electrophoresis and densitometric measurements.
Functional assay
4BB™ QualiPhi® DNA Polymerase has been tested for exponential amplification of 10pg and 1ng of high molecular weight human genomic DNA. In the absence of DNA input (non-template control, NTC) no background amplification was exhibited under similar conditions.
Shipping and Storage
4BB™ QualiPhi® DNA polymerase is shipped on dry ice. On receipt, the kit should be stored immediately at -20oC in a non frost-free (constant temperature) freezer. When stored correctly, the product can be kept for a minimum of six months after shipping without displaying any reduction in performance. For longer periods, store the kit at -80oC. Avoid repeated freeze-thaw cycles.
