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 by acting as a primer for the viral DNA polymerase.
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.[citation needed]
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]Deoxyribonucleosidetriphosphate cleavage that occurs as part of the polymerization 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 synthesized strand.[5] The exonuclease activity of the enzyme is, like its polymerization 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 polymerization 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]
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
^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.
PMID3803926.
^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.
PMID2121621.
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.
PMID20926164.
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.
hdl:10486/709525.
PMID19883660.
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.
hdl:10486/709542.
PMID19576228.
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.
PMID19375325.
Silander K, Saarela J (2008). "Whole Genome Amplification with Phi29 DNA Polymerase to Enable Genetic or Genomic Analysis of Samples of Low DNA Yield". Genomics Protocols. Methods in Molecular Biology. Vol. 439. pp. 1–18.
doi:
10.1007/978-1-59745-188-8_1.
ISBN978-1-58829-871-3.
PMID18370092.
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.
PMID17226067.
S2CID12464800.
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.
PMID17174409.
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.
PMID16023891.
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.
PMID15777661.
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.
PMID15721296.
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.
PMID15168072.
S2CID11041367.
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.
PMID15033354.
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.
PMID14738990.
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.
PMID14672657.
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.
PMID12473453.
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.
PMID12083397.
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.
PMID12054770.
de Vega M, Blanco L, Salas M (1999). "Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage phi29 DNA polymerase". J Mol Biol. 292 (1): 39–51.
doi:
10.1006/jmbi.1999.3052.
PMID10493855.
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.
PMID10388570.
Truniger V, Blanco L, Salas M (1999). "Role of the "YxGG/A" motif of Phi29 DNA polymerase in protein-primed replication". J Mol Biol. 286 (1): 57–69.
doi:
10.1006/jmbi.1998.2477.
PMID9931249.
de Vega M, Lázaro JM, Salas M, Blanco L (1998). "Mutational analysis of phi29 DNA polymerase residues acting as ssDNA ligands for 3'-5' exonucleolysis". J Mol Biol. 279 (4): 807–22.
doi:
10.1006/jmbi.1998.1805.
PMID9642062.
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 by acting as a primer for the viral DNA polymerase.
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.[citation needed]
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]Deoxyribonucleosidetriphosphate cleavage that occurs as part of the polymerization 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 synthesized strand.[5] The exonuclease activity of the enzyme is, like its polymerization 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 polymerization 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]
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
^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.
PMID3803926.
^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.
PMID2121621.
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.
PMID20926164.
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.
hdl:10486/709525.
PMID19883660.
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.
hdl:10486/709542.
PMID19576228.
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.
PMID19375325.
Silander K, Saarela J (2008). "Whole Genome Amplification with Phi29 DNA Polymerase to Enable Genetic or Genomic Analysis of Samples of Low DNA Yield". Genomics Protocols. Methods in Molecular Biology. Vol. 439. pp. 1–18.
doi:
10.1007/978-1-59745-188-8_1.
ISBN978-1-58829-871-3.
PMID18370092.
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.
PMID17226067.
S2CID12464800.
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.
PMID17174409.
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.
PMID16023891.
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.
PMID15777661.
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.
PMID15721296.
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.
PMID15168072.
S2CID11041367.
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.
PMID15033354.
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.
PMID14738990.
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.
PMID14672657.
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.
PMID12473453.
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.
PMID12083397.
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.
PMID12054770.
de Vega M, Blanco L, Salas M (1999). "Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage phi29 DNA polymerase". J Mol Biol. 292 (1): 39–51.
doi:
10.1006/jmbi.1999.3052.
PMID10493855.
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.
PMID10388570.
Truniger V, Blanco L, Salas M (1999). "Role of the "YxGG/A" motif of Phi29 DNA polymerase in protein-primed replication". J Mol Biol. 286 (1): 57–69.
doi:
10.1006/jmbi.1998.2477.
PMID9931249.
de Vega M, Lázaro JM, Salas M, Blanco L (1998). "Mutational analysis of phi29 DNA polymerase residues acting as ssDNA ligands for 3'-5' exonucleolysis". J Mol Biol. 279 (4): 807–22.
doi:
10.1006/jmbi.1998.1805.
PMID9642062.