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phi29 DNA Polymerase

Recombinant enzyme Thermal inactivation at 65°C in 10 min

phi29 DNA Polymerase is a highly processive polymerase with strong strand displacement activity that allows for highly efficient isothermal DNA amplification.
  
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Thermo Scientific phi29 DNA Polymerase is a highly processive polymerase (up to more than 70 kb) featuring strong strand displacement activity, which allows for highly efficient isothermal DNA amplification (see Reference 1). phi29 DNA Polymerase also possesses a 3'→5' exonuclease (proofreading) activity acting preferentially on single-stranded DNA (see Reference 2) or RNA (see Reference 3). Therefore 3'-modified primers are highly recommended (see Reference 4).

Highlights

  • Highest processivity and strand displacement activity among known DNA polymerases – more than 70 kb long DNA stretches can be synthesized (see Reference 1)
  • Highly accurate DNA synthesis (see Reference 5)
  • Extremely high yields of amplified DNA even from minute amounts of template
  • Amplification products can be directly used in downstream applications (PCR, restriction digestion, SNP genotyping, etc.)

Applications

  • Rolling circle amplification (RCA) (see Reference 6): generation of periodic DNA nanotemplates (see Reference 7)
  • Multiple displacement amplification (MDA) (see Reference 8)
  • Unbiased amplification of whole genome (WGA, see Figure 1 in Supporting Data):
    • amplification of DNA for SNP (see Reference 9) and STR (see Reference 10) detection
    • cell-free amplification of DNA from single cells (see References 11, 12)
    • pathogenic organisms or metagenomes (see Reference 13)
    • amplification of DNA from filter paper blood spot samples (see Reference 14)
     
  • DNA template preparation for sequencing
  • Protein-primed DNA amplification (see Reference 15)
  • In situ genotyping with padlock probes (see Reference 17)
  • Recombination based-cloning (see Reference 18)
  • Cell-free cloning of lethal DNA (see Reference 19)
  • RNA-primed DNA amplification (see Reference 16)

Note

Addition of Pyrophosphatase to the reaction mixture with phi29 DNA Polymerase may enhance DNA synthesis (see Reference 8).

Use of this enzyme in certain applications may be covered by patents and may require a license.

  
Storage Condition-20 C
HazardousNo
10× Reaction Buffer330 mM Tris-acetate (pH 7.9 at 37°C), 100 mM Mg-acetate, 660 mM K-acetate, 1% (v/v) Tween 20, 10 mM DTT.
Definition of Activity Unit
  • One unit of the enzyme catalyzes the incorporation of 0.5 pmol of dCMP into a polynucleotide fraction (adsorbed on DE-81) in 10 min at 30°C.
  • Enzyme activity is assayed in the following mixture: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 0.01 mg/mL lambda DNA/HindIII, 0.2 µM dCTP, including [3H]-dCTP, 0.2 mM dATP, 0.2 mM dGTP, and 0.2 mM dTTP.
InactivationInactivated by heating at 65°C for 10 min.
InhibitionInhibitors: aphidicolin, N2-(p-n-butylphenyl)-dGTP (BuPdGTP), 2-(p-n-butylanilino)-dATP (BuAdATP) (20).
Molecular Weight66.7 kDa monomer
Quality ControlThe absence of endodeoxyribonucleases confirmed by appropriate quality tests.
SourceE.coli cells with a cloned gene 2 of Bacillus subtilis phage phi29.
Specific Activity100,000 U/mg.
Storage BufferThe enzyme is supplied in 50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM DTT, 100 mM KCl, 0.5% (v/v) Nonidet P40, 0.5% (v/v) Tween 20, and 50% (v/v) glycerol.
Unbiased amplification of human genomic DNA with phi29 DNA Polymerase

Unbiased amplification of human genomic DNA with phi29 DNA Polymerase

Unbiased amplification of human genomic DNA with phi29 DNA Polymerase

Unbiased amplification of human genomic DNA with phi29 DNA Polymerase.
100 copies (0.33 ng) of human genomic DNA were amplified with phi29 DNA Polymerase according to the procedure of Dean et al., 2002 (see Reference 9). Radioactive [α-33P]-dATP incorporation into the DE-81 adsorbable form was calculated to quantify amplification of total DNA. Amplification efficiency of specific genes located in different chromosomes was determined by real-time PCR using the ABI 7700.


References

  1. L. Blanco et al., Highly Efficient DNA Synthesis by the phage phi29 DNA Polymerase. Symmetrical mode of DNA replication. J. Biol. Chem. 264, 8935-8940 (1989).
  2. C. Garmendia et al., The bacteriophage phi29 DNA polymerase, a proofreading enzyme. J. Biol. Chem. 267, 2594-2599 (1992).
  3. A. Lagunavicius et al., Duality of polynucleotide substrates for phi29 DNA polymerase: 3’=>5’ RNase activity of the enzyme. RNA. 14, 503-513 (2008).
  4. A. Skerra, Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity. Nucleic Acids Res. 20, 3551-3554 (1992).
  5. J. A. Esteban et al., Fidelity of phi29 DNA polymerase. J. Biol. Chem. 268(4), 2719-2726 (1993).
  6. P. M. Lizardi et al., Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat. Genet. 19, 225-232 (1998).
  7. F. C. Simmel et al., Periodic DNA nanotemplates synthesized by rolling circle amplification. Nano Letters. 4, 719-722 (2005).
  8. F. B. Dean et al., Comprehensive human genome amplification using multiple displacement amplification. Proc. Natl. Acad. Sci. USA. 99, 5261-5266 (2002).
  9. L. Wojnowski et al., Genome-wide single-nucleotide polymorphism arrays demonstrate high fidelity of multiple displacement-based whole-genome amplification. Electrophoresis. 26, 710–715 (2005).
  10. M. R. James et al., Evaluation of multiple displacement amplification in a 5 cM STR genome-wide scan. Nucleic Acids Res. 13, e1191 (2005).
  11. V. Gadkar, M. C. Rillig, Suitability of genomic DNA synthesized by strand displacement amplification (SDA) for AFLP analysis, genotyping single spores of arbuscular mycorrhizal (AM) fungi. J. Microbiol. Methods. 11(63), 157-164 (2005).
  12. V. Sgaramella et al., Towards the analysis of the genomes of single cells, Further characterisation of the multiple displacement amplification. Gene. 372, 1–7 (2006).
  13. T. Matsunaga et al., Whole-metagenome amplification of a microbial community associated with scleractinian coral by multiple displacement amplification using phi29 polymerase. Environmental Microbiology. 8, 1155–1163 (2006).
  14. P. S. Andersen et al., Whole genome amplification on DNA from filter paper blood spot samples, an evaluation of selected systems. Genetic Testing. 11(1), 65-71 (Spring 2007).
  15. L. Blanco et al., Terminal protein – primed DNA amplification. Proc. Natl. Acad. Sci. USA. 91(25), 12198-12202 (1994).
  16. A. Lagunavicius et al., Novel application of phi29 DNA polymerase: RNA detection and analysis in vitro and in situ by target RNA-primed RCA. RNA. 15(5), 765-771 (May 2009).
  17. C. Larsson et al., In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes. Nature Methods. 1(3), 227-232 (December 2004).
  18. M. B. Jakov, P. D. Kassner, Multiple displacement amplification products are compatible with recombination-based cloning. BioTechniques. 42(6), 706-708 (June 2007).
  19. C. A. Hutchison et al., Cell-free cloning using phi29 DNA polymerase. Proc. Natl. Acad. Sci. U S A. 102(48), 17332-17336 (29 November 2005).
  20. L. Blanco, M. Salas, Effect of aphidicolin and nucleotide analogs of on the phage phi29 DNA polymerase. Virology. 153(2), 179-187 (September 1986).
  21. EPO Patent No 0862656B1.
  22. US Patent No 5,001,050.

Protocols and Product Manuals

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