Phi29 Polymerase

The phi29 polymerase is the only replicative polymerase required for the efficient replication of the Bacillus subtilis phage phi29 DNA. This is made possible by the highly processivity of the enzyme without the requirements of any other replicative protein and exceptional strand displacement coupled to the polymerization process. This makes the phi29 polymerase very efficient for isothermal DNA amplification as the enzyme is capable of amplification of up to 70 kb insertions per binding event. The enzyme also possesses a 3’→5’ proofreading exonuclease activity, acting preferentially on ssDNA or RNA. These properties make the phi29 polymerase an ideal tool to achieve isothermal strand displacement amplification.

SPECIFICATION
• VPCIR ™ phi29 polymerase holds marked processivity and strand displacement activity allowing for long DNA stretches to be synthesized (>70kb).
• High-fidelity polymerase – possesses a 3’-5’ exonuclease (proofreading) activity acting preferentially on ssDNA or RNA.
• High yields of amplified DNA even from small template amounts
• Isothermal polymerase – no need for thermal cycling.

APPLICATIONS
• Isothermal rolling circle amplification (RCA)
• Isothermal amplification for sequencing and cloning
• In situ genotyping with padlock probe
• Multiple displacement amplification (MDA)
• Unbiased whole genome amplification (WGA)
• Amplification of DNA for SNP and STR detection
• Cell-free amplification of DNA from single cells
• Pathogenic organisms or metagenomes
• Amplification of DNA from filter paper spot samples
• Amplification of DNA from environmental samples
• DNA template preparation for sequencing
• Protein-primed DNA amplification
• Recombination based-cloning
• Cell-free cloning of lethal DNA
• RNA-primed DNA amplification
• Downstream applications such as PCR, restriction digestion, SNP genotyping, etc.

The phi29 enzyme is supplied together with:
• Dilution Buffer: 50mM Tris-HCL pH 7.5; 1mM DTT; 50% glycerol; 0.5%Tween 20; 0.5% NP40; 500mM NaCl.
• Reaction Buffer 10x: 330 mM Tris-acetate; 100 mM Magnesium-acetate, 660 mM Potassium-acetate; 1% Tween20; 2mM DTT (to be added fresh).
• 5 M DTT
• Nucleases-free water

UNIT DEFINITION
Phi29 unit has been determined by comparison with Thermofisher (EP0091) phi29 polymerase where 1 unit is defined as the amount of the enzyme that catalyzes the incorporation of 0.5 pmol of dCMP into a polynucleotide fraction in 10 min at 30°C

Recombinant GST-tagged phi29 enzyme is prepared by overexpressing the enzyme in E. coli followed by purification by affinity chromatography

The enzyme is shipped cooled by gel packs and must be stored at -18°C to -20°C upon arrival. The temperature must be controlled to avoid that the enzyme preparation solidifies. This would destroy the enzyme integrity and cause a drop of the phi29 activity. The enzyme has a lifetime of 12 months when stored under optimal conditions

Certificate of analysis of VPCIR™ phi29 DNA Polymerase

Download the Safety Data Sheet

1. Blanco, L.; Bernad, A.; Lázaro, J.M.; Martín, G.; Garmendia, C.; Salas, M. Highly Efficient DNA Synthesis by the Phage ϕ 29 DNA Polymerase. J. Biol. Chem. 1989, 264, 8935–8940, doi:10.1016/s0021-9258(18)81883-x.

2. Yokouchi, H.; Fukuoka, Y.; Mukoyama, D.; Calugay, R.; Takeyama, H.; Matsunaga, T. Whole-metagenome amplification of a microbial community associated with scleractinian coral by multiple displacement amplification using j 29 polymerase. 2006, 8, 1155–1163, doi:10.1111/j.1462-2920.2006.01005.x.

3. Larsson, C.; Koch, J.; Nygren, A.; Janssen, G.; Raap, A.K.; Landegren, U. In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes. 2004, 1, 1–6, doi:10.1038/NMETH723.

4. Salas, M.; Blanco, L.; Lázaro, J.M.; De Vega, M. The bacteriophage φ29 DNA polymerase. IUBMB Life 2008, 60, 82–85, doi:10.1002/iub.19.