Φ29 DNA polymerase

Φ29 DNA polymerase is an enzyme from the bacteriophage Φ29. It is being increasingly used in molecular biology for multiple displacement DNA amplification procedures, and has a number of features that make it particularly suitable for this application.

Φ29 DNA replication

Φ29 is a bacteriophage of Bacillus subtilis with a sequenced, linear, 19,285 base pair DNA genome.[1] Each 5' end is covalently linked to a terminal protein, which is essential in the replication process. A symmetrical mode of replication has been suggested, whereby protein-primed initiation occurs non-simultaneously from either end of the chromosome; this involves two replication origins and two distinct polymerase monomers. Synthesis is continual and involves a strand displacement mechanism. This was demonstrated by the ability of the enzyme to continue to copy the singly primed circular genome of the M13 phage more than tenfold in a single strand (over 70kb in a single strand).[2] In vitro experiments have shown that Φ29 replication can proceed to completion with the sole phage protein requirements of the polymerase and the terminal protein.[2] The polymerase catalyses the formation of the initiation complex between the terminal protein and the chromosome ends at an adenine residue. From here, continual synthesis can occur.

The polymerase

The polymerase is a monomeric protein with two distinct functional domains. Site-directed mutagenesis experiments support the proposition that this protein displays a structural and functional similarity to the Klenow fragment of the Escherichia coli Polymerase I enzyme;[3] it comprises a C-terminal polymerase domain and a spatially separated N-terminal domain with a 3'-5' exonuclease activity.

The isolated enzyme has no intrinsic helicase activity, but may carry out an equivalent function by way of its strong binding to single stranded DNA, particularly in preference to double stranded nucleic acid. This is the property of this enzyme that makes is favorably applicable to Multiple Displacement Amplification. The enzyme facilitates the "debranching" of double stranded DNA.[2] Deoxyribonucleoside triphosphate cleavage that occurs as part of the polymerisation process probably supplies the energy required for this unwinding mechanism.[4] The continuous nature of strand synthesis (compared to the asymmetric synthesis seen in other organisms) probably contributes to this enhanced processivity. Proofreading activity conferred by the exonuclease domain was demonstrated by showing the preferential excision of a mismatched nucleotide from the 3' terminus of the newly synthesised strand.[5] The exonuclease activity of the enzyme is, like its polymerisation activity, highly processive and can degrade single stranded oligonucleotides without dissociation. Co-operation or a 'delicate competition' between these two functional domains is essential, so as to ensure accurate elongation at an optimal rate. The exonuclease activity of the enzyme does impede its polymerisation capacity; inactivation of the exonuclease activity by site-directed mutagenesis meant that a 350 fold lower dNTP concentration was required to achieve the same rates of primer elongation seen in the wild type enzyme.[5]

Whole genome amplification

Φ29 polymerase enzyme is already used in multiple displacement amplification (MDA) procedures (including in a number of commercial kits) whereby fragments tens of kilobases in length can be produced from non-specific hexameric primers annealing at intervals along the genome. The enzyme has many desirable properties that make it appropriate for whole genome amplification (WGA) by this method.[6]

  • High processivity.[2]
  • Proofreading activity.[5] It is believed to be 1 or 2 orders of magnitude less error prone than Taq polymerase.[7]
  • Generates large fragments, over 10kb.
  • Produces more DNA than PCR-based methods, by about an order of magnitude.[8]
  • Requires minimal amount of template; 10 ng suffices.
  • Novel replication mechanism; multiple-strand displacement amplification.
    • Random primers (hexamers) can be used, no need to design specific primers/target specific regions.
    • No need for thermal cycling.
  • Good coverage and a reduced amplification bias when compared to PCR-based approaches. There is speculation that it is the least biased of the WGA methods in use.[8]

References

  1. ↑ Vlcek C, Paces V (1986). "Nucleotide sequence of the late region of Bacillus phage Φ29 completes the 19,285-bp sequence of Φ29 genome. Comparison with the homologous sequence of phage PZA". Gene. 46 (2–3): 215–25. doi:10.1016/0378-1119(86)90406-3. PMID 3803926.
  2. 1 2 3 4 Blanco L, Bernad A, Lázaro JM, Martín G, Garmendia C, Salas M (May 1989). "Highly efficient DNA synthesis by the phage Φ29 DNA polymerase. Symmetrical mode of DNA replication". J. Biol. Chem. 264 (15): 8935–40. PMID 2498321.
  3. ↑ Bernad A, Blanco L, Salas M (September 1990). "Site-directed mutagenesis of the YCDTDS amino acid motif of the phi 29 DNA polymerase". Gene. 94 (1): 45–51. doi:10.1016/0378-1119(90)90466-5. PMID 2121621.
  4. ↑ Alberts B, Sternglanz R (October 1977). "Recent excitement in the DNA replication problem". Nature. 269 (5630): 655–61. doi:10.1038/269655a0. PMID 201853.
  5. 1 2 3 Garmendia C, Bernad A, Esteban JA, Blanco L, Salas M (February 1992). "The bacteriophage phi 29 DNA polymerase, a proofreading enzyme". J. Biol. Chem. 267 (4): 2594–9. PMID 1733957.
  6. ↑ Alsmadi O, Alkayal F, Monies D, Meyer BF (2009). "Specific and complete human genome amplification with improved yield achieved by phi29 DNA polymerase and a novel primer at elevated temperature". BMC Res Notes. 2: 48. doi:10.1186/1756-0500-2-48. PMC 2663774. PMID 19309528.
  7. ↑ Pugh TJ, Delaney AD, Farnoud N, et al. (August 2008). "Impact of whole genome amplification on analysis of copy number variants". Nucleic Acids Res. 36 (13): e80. doi:10.1093/nar/gkn378. PMC 2490749. PMID 18559357.
  8. 1 2 Pinard R, de Winter A, Sarkis GJ, et al. (2006). "Assessment of whole genome amplification-induced bias through high-throughput, massively parallel whole genome sequencing". BMC Genomics. 7: 216. doi:10.1186/1471-2164-7-216. PMC 1560136. PMID 16928277.

Further reading


  • Linck L, Resch-Genger U (2010). "Identification of efficient fluorophores for the direct labeling of DNA via rolling circle amplification (RCA) polymerase φ29". Eur J Med Chem. 45 (12): 5561–6. doi:10.1016/j.ejmech.2010.09.005. PMID 20926164.
  • de Vega M, Lázaro JM, Mencía M, Blanco L, Salas M (2010). "Improvement of φ29 DNA polymerase amplification performance by fusion of DNA binding motifs". Proc Natl Acad Sci U S A. 107 (38): 16506–11. doi:10.1073/pnas.1011428107. PMC 2944734. PMID 20823261.
  • Pérez-Arnaiz P, Lázaro JM, Salas M, de Vega M (2010). "phi29 DNA polymerase active site: role of residue Val250 as metal-dNTP complex ligand and in protein-primed initiation". J Mol Biol. 395 (2): 223–33. doi:10.1016/j.jmb.2009.10.061. PMID 19883660.
  • Pérez-Arnaiz P, Lázaro JM, Salas M, de Vega M (2009). "Functional importance of bacteriophage phi29 DNA polymerase residue Tyr148 in primer-terminus stabilisation at the 3'-5' exonuclease active site". J Mol Biol. 391 (5): 797–807. doi:10.1016/j.jmb.2009.06.068. PMID 19576228.
  • Johne R, Müller H, Rector A, van Ranst M, Stevens H (2009). "Rolling-circle amplification of viral DNA genomes using phi29 polymerase". Trends Microbiol. 17 (5): 205–11. doi:10.1016/j.tim.2009.02.004. PMID 19375325.
  • Lagunavicius A, Merkiene E, Kiveryte Z, Savaneviciute A, Zimbaite-Ruskuliene V, Radzvilavicius T, Janulaitis A (2009). "Novel application of Phi29 DNA polymerase: RNA detection and analysis in vitro and in situ by target RNA-primed RCA". RNA. 15 (5): 765–71. doi:10.1261/rna.1279909. PMC 2673074. PMID 19244362.
  • Rodríguez I, Lázaro JM, Salas M, de Vega M (2009). "Involvement of the TPR2 subdomain movement in the activities of phi29 DNA polymerase". Nucleic Acids Res. 37 (1): 193–203. doi:10.1093/nar/gkn928. PMC 2615600. PMID 19033368.
  • Sahu S, LaBean TH, Reif JH (2008). "A DNA nanotransport device powered by polymerase phi29". Nano Lett. 8 (11): 3870–8. doi:10.1021/nl802294d. PMID 18939810.
  • Xu Y, Gao S, Bruno JF, Luft BJ, Dunn JJ (2008). "Rapid detection and identification of a pathogen's DNA using Phi29 DNA polymerase". Biochem. Biophys. Res. Commun. 375 (4): 522–5. doi:10.1016/j.bbrc.2008.08.082. PMC 2900840. PMID 18755142.
  • Kumar G, Garnova E, Reagin M, Vidali A (2008). "Improved multiple displacement amplification with phi29 DNA polymerase for genotyping of single human cells". Biotechniques. 44 (7): 879–90. doi:10.2144/000112755. PMID 18533898.
  • Salas M, Blanco L, Lázaro JM, de Vega M (2008). "The bacteriophage phi29 DNA polymerase". IUBMB Life. 60 (1): 82–5. doi:10.1002/iub.19. PMID 18379997.
  • Silander K, Saarela J (2008). "Whole genome amplification with Phi29 DNA polymerase to enable genetic or genomic analysis of samples of low DNA yield". Methods Mol Biol. 439: 1–18. doi:10.1007/978-1-59745-188-8_1. PMID 18370092.
  • Lagunavicius A, Kiveryte Z, Zimbaite-Ruskuliene V, Radzvilavicius T, Janulaitis A (2008). "Duality of polynucleotide substrates for Phi29 DNA polymerase: 3'-->5' RNase activity of the enzyme". RNA. 14 (3): 503–13. doi:10.1261/rna.622108. PMC 2248250. PMID 18230765.
  • Pérez-Arnaiz P, Longás E, Villar L, Lázaro JM, Salas M, de Vega M (2007). "Involvement of phage phi29 DNA polymerase and terminal protein subdomains in conferring specificity during initiation of protein-primed DNA replication". Nucleic Acids Res. 35 (21): 7061–73. doi:10.1093/nar/gkm749. PMC 2175359. PMID 17913744.
  • Berman AJ, Kamtekar S, Goodman JL, Lázaro JM, de Vega M, Blanco L, Salas M, Steitz TA (2007). "Structures of phi29 DNA polymerase complexed with substrate: the mechanism of translocation in B-family polymerases". EMBO J. 26 (14): 3494–505. doi:10.1038/sj.emboj.7601780. PMC 1933411. PMID 17611604.
  • Knierim D, Maiss E (2007). "Application of Phi29 DNA polymerase in identification and full-length clone inoculation of tomato yellow leaf curl Thailand virus and tobacco leaf curl Thailand virus". Arch Virol. 152 (5): 941–54. doi:10.1007/s00705-006-0914-9. PMID 17226067.
  • Owor BE, Shepherd DN, Taylor NJ, Edema R, Monjane AL, Thomson JA, Martin DP, Varsani A (2007). "Successful application of FTA Classic Card technology and use of bacteriophage phi29 DNA polymerase for large-scale field sampling and cloning of complete maize streak virus genomes". J Virol Methods. 140 (1–2): 100–5. doi:10.1016/j.jviromet.2006.11.004. PMID 17174409.
  • Sato M, Ohtsuka M, Ohmi Y (2004). "Repeated GenomiPhi, phi29 DNA polymerase-based rolling circle amplification, is useful for generation of large amounts of plasmid DNA". Nucleic Acids Symp Ser (Oxf). 48 (48): 147–8. doi:10.1093/nass/48.1.147. PMID 17150521.
  • Pérez-Arnaiz P, Lázaro JM, Salas M, de Vega M (2006). "Involvement of phi29 DNA polymerase thumb subdomain in the proper coordination of synthesis and degradation during DNA replication". Nucleic Acids Res. 34 (10): 3107–15. doi:10.1093/nar/gkl402. PMC 1475753. PMID 16757576.
  • Kamtekar S, Berman AJ, Wang J, Lázaro JM, de Vega M, Blanco L, Salas M, Steitz TA (2006). "The phi29 DNA polymerase:protein-primer structure suggests a model for the initiation to elongation transition". EMBO J. 25 (6): 1335–43. doi:10.1038/sj.emboj.7601027. PMC 1422159. PMID 16511564.
  • Hutchison CA, Smith HO, Pfannkoch C, Venter JC (2005). "Cell-free cloning using phi29 DNA polymerase". Proc Natl Acad Sci U S A. 102 (48): 17332–6. doi:10.1073/pnas.0508809102. PMC 1283157. PMID 16286637.
  • Sato M, Ohtsuka M, Ohmi Y (2005). "Usefulness of repeated GenomiPhi, a phi29 DNA polymerase-based rolling circle amplification kit, for generation of large amounts of plasmid DNA". Biomol Eng. 22 (4): 129–32. doi:10.1016/j.bioeng.2005.05.001. PMID 16023891.
  • Rodríguez I, Lázaro JM, Blanco L, Kamtekar S, Berman AJ, Wang J, Steitz TA, Salas M, de Vega M (2005). "A specific subdomain in phi29 DNA polymerase confers both processivity and strand-displacement capacity". Proc Natl Acad Sci U S A. 102 (18): 6407–12. doi:10.1073/pnas.0500597102. PMC 1088371. PMID 15845765.
  • Truniger V, Bonnin A, Lázaro JM, de Vega M, Salas M (2005). "Involvement of the "linker" region between the exonuclease and polymerization domains of phi29 DNA polymerase in DNA and TP binding". Gene. 348: 89–99. doi:10.1016/j.gene.2004.12.041. PMID 15777661.
  • Umetani N, de Maat MF, Mori T, Takeuchi H, Hoon DS (2005). "Synthesis of universal unmethylated control DNA by nested whole genome amplification with phi29 DNA polymerase". Biochem. Biophys. Res. Commun. 329 (1): 219–23. doi:10.1016/j.bbrc.2005.01.088. PMID 15721296.
  • Gadkar V, Rillig MC (2005). "Application of Phi29 DNA polymerase mediated whole genome amplification on single spores of arbuscular mycorrhizal (AM) fungi". FEMS Microbiol Lett. 242 (1): 65–71. doi:10.1016/j.femsle.2004.10.041. PMID 15621421.
  • Kamtekar S, Berman AJ, Wang J, Lázaro JM, de Vega M, Blanco L, Salas M, Steitz TA (2004). "Insights into strand displacement and processivity from the crystal structure of the protein-primed DNA polymerase of bacteriophage phi29". Mol Cell. 16 (4): 609–18. doi:10.1016/j.molcel.2004.10.019. PMID 15546620.
  • Adachi E, Shimamura K, Wakamatsu S, Kodama H (2004). "Amplification of plant genomic DNA by Phi29 DNA polymerase for use in physical mapping of the hypermethylated genomic region". Plant Cell Rep. 23 (3): 144–7. doi:10.1007/s00299-004-0806-y. PMID 15168072.
  • Rodríguez I, Lázaro JM, Salas M, De Vega M (2004). "phi29 DNA polymerase-terminal protein interaction. Involvement of residues specifically conserved among protein-primed DNA polymerases". J Mol Biol. 337 (4): 829–41. doi:10.1016/j.jmb.2004.02.018. PMID 15033354.
  • Inoue-Nagata AK, Albuquerque LC, Rocha WB, Nagata T (2004). "A simple method for cloning the complete begomovirus genome using the bacteriophage phi29 DNA polymerase". J Virol Methods. 116 (2): 209–11. doi:10.1016/j.jviromet.2003.11.015. PMID 14738990.
  • Truniger V, Lázaro JM, Salas M (2004). "Function of the C-terminus of phi29 DNA polymerase in DNA and terminal protein binding". Nucleic Acids Res. 32 (1): 361–70. doi:10.1093/nar/gkh184. PMC 373294. PMID 14729920.
  • Truniger V, Lázaro JM, Salas M (2004). "Two positively charged residues of phi29 DNA polymerase, conserved in protein-primed DNA polymerases, are involved in stabilisation of the incoming nucleotide". J Mol Biol. 335 (2): 481–94. doi:10.1016/j.jmb.2003.10.024. PMID 14672657.
  • Rodríguez I, Lázaro JM, Salas M, de Vega M (2003). "phi29 DNA polymerase residue Phe128 of the highly conserved (S/T)Lx(2)h motif is required for a stable and functional interaction with the terminal protein". J Mol Biol. 325 (1): 85–97. doi:10.1016/S0022-2836(02)01130-0. PMID 12473453.
  • Nelson JR, Cai YC, Giesler TL, Farchaus JW, Sundaram ST, Ortiz-Rivera M, Hosta LP, Hewitt PL, Mamone JA, Palaniappan C, Fuller CW (2002). "TempliPhi, phi29 DNA polymerase based rolling circle amplification of templates for DNA sequencing". Biotechniques. Suppl: 44–7. PMID 12083397.
  • Truniger V, Lázaro JM, Blanco L, Salas M (2002). "A highly conserved lysine residue in phi29 DNA polymerase is important for correct binding of the templating nucleotide during initiation of phi29 DNA replication". J Mol Biol. 318 (1): 83–96. doi:10.1016/S0022-2836(02)00022-0. PMID 12054770.
  • Truniger V, Lázaro JM, Esteban FJ, Blanco L, Salas M (2002). "A positively charged residue of phi29 DNA polymerase, highly conserved in DNA polymerases from families A and B, is involved in binding the incoming nucleotide". Nucleic Acids Res. 30 (7): 1483–92. doi:10.1093/nar/30.7.1483. PMC 101840. PMID 11917008.
  • Eisenbrandt R, Lázaro JM, Salas M, de Vega M (2002). "Phi29 DNA polymerase residues Tyr59, His61 and Phe69 of the highly conserved ExoII motif are essential for interaction with the terminal protein". Nucleic Acids Res. 30 (6): 1379–86. doi:10.1093/nar/30.6.1379. PMC 101362. PMID 11884636.
  • Elías-Arnanz M, Salas M (1999). "Resolution of head-on collisions between the transcription machinery and bacteriophage phi29 DNA polymerase is dependent on RNA polymerase translocation". EMBO J. 18 (20): 5675–82. doi:10.1093/emboj/18.20.5675. PMC 1171634. PMID 10523310.
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  • Bonnin A, Lázaro JM, Blanco L, Salas M (1999). "A single tyrosine prevents insertion of ribonucleotides in the eukaryotic-type phi29 DNA polymerase". J Mol Biol. 290 (1): 241–51. doi:10.1006/jmbi.1999.2900. PMID 10388570.
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