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Figure 1. 100pg of pUC19 was amplified using either a low concentration or high concentration stock of PD-Phi29. The results were compared to the current market best and quantified using a dsDNA dye. The dsDNA production time-course (left graph) was performed at 42°C.

Figure 2. Whole Genome Amplification was performed on a mix of three bacterial genomes covering a range of GC content, and GC bias of MDA measured via PCR-free Illumina sequencing of the resulting product. Grey bars show the GC distribution of Illumina sequencing data of the input genomic DNA (no WGA), black MDA performed by wild-type phi29, blue using a kit purchased from the market leader, and orange using PD-phi29. The orange line (PD-phi29) is closest to the grey bars, meaning that PD-phi29 has the lowest GC bias. Left panel shows histograms, and the right panel shows the deviation of each WGA sample from the unamplified input DNA. Earth Mover's distance was used to quantify the difference between samples.

Figure 3. Single bacterial colonies were picked, lysed using a thermocycler, and RCA reactions were carried out with the market leading enzyme according to their protocol (grey bar), or with PD-phi29 RCA Kit at the shown temperature, for 1 hour. The amplification products debranched with T7 endonuclease and sequenced using Nanopore. PD-Phi29 has higher yield than phi29-XT, and a higher success rate for correct and complete full-plasmid sequencing.

Figure 4. A bacterial colony was diluted into water (standard direct-from-colony protocol) or into the reaction buffer with dNTPs and hexamers (PD-phi29 protocol) at a variety of cell densities (X axis), lysed using a thermocycler, and RCA reactions were carried out using the PD-phi29 RCA Kit with either low or high enzyme concentration tubes of PD-phi29. The amplification products debranched with T7 endonuclease and sequenced using Nanopore. The PD-phi29 protocol and high enzyme concentration improve success for direct-fromcolony plasmid sequencing.

Figure 5. RCA reactions were carried out with the PD-Phi29-XT RCA Kit at 42°C for 1 hour (low enzyme concentration) in the absence of template, or with 1 fg - 10 pg of pUC19 plasmid template. The RCA products were then digested with AclI, which creates 373 bp and 2313 bp fragments. The digests were run on a 1% agarose gel and later stained with SYBR Gold nucleic acid stain.

Figure 6. PD-Phi29 in storage buffer was aliquoted into a thin-walled PCR tube and transferred from -20°C for 10 minutes to room temperature where it was allowed to thaw for five minutes. Eleven freeze-thaw cycles were performed on the original enzyme stock and a small aliquot was taken after each cycle and used to amplify 10pg of pUC19. RCA reactions were carried out in a QuantStudio qPCR machine, and dsDNA was quantified every five minutes in the qPCR machine.

PD-Phi29 Engineered phi29 DNA Polymerase

PD-Phi29 Engineered phi29 DNA Polymerase

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E001504996PAN

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PD-Phi29

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Overview

We offer engineered multiple phi29 DNA polymerase variants with novel properties that significantly improve the efficiency and versatility of DNA amplification techniques. Identified through Pando’s technology, these mutations offer several advantages over wild-type Phi29 polymerase, including:
• Increased activity
• Increased thermostability
• Increased manufacturability

Features
• PD-Phi29 is active up to 42°C and creates more DNA faster than the market leader.
• PD-Phi29 has a higher success rate for correct and complete full-plasmid sequencing than the market best.
• PD-Phi29 protocol and high enzyme concentration improve success for direct-from-colony plasmid sequencing.
• PD-Phi29 supports robust and specific amplification of as little as 10 fg of purified plasmid DNA in just one hour.
• PD-Phi29 is stable over more than ten freeze-thaw cycles.

Please contact us at for specific academic pricing.

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Replicative DNA polymerases

* For Research Use Only.

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