Φ29 DNA polymerase
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Φ29 DNA polymerase is an enzyme from the bacteriophage Φ29. It is being increasingly used in
molecular biology Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...
for multiple displacement DNA amplification procedures, and has a number of features that make it particularly suitable for this application. It was discovered and characterised by Spanish scientist Margarita Salas.


Φ29 DNA replication

Φ29 is a bacteriophage of ''
Bacillus subtilis ''Bacillus subtilis'', known also as the hay bacillus or grass bacillus, is a Gram-positive, catalase-positive bacterium, found in soil and the gastrointestinal tract of ruminants, humans and marine sponges. As a member of the genus ''Bacillus ...
'' with a sequenced, linear, 19,285 base pair DNA genome. 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 M13 is one of the Ff phages (fd and f1 are others), a member of the family filamentous bacteriophage ( inovirus). Ff phages are composed of circular single-stranded DNA ( ssDNA), which in the case of the m13 phage is 6407 nucleotides long and ...
more than tenfold in a single strand (over 70kb in a single strand). 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. 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 In chemistry, a monomer ( ; ''mono-'', "one" + '' -mer'', "part") is a molecule that can react together with other monomer molecules to form a larger polymer chain or three-dimensional network in a process called polymerization. Classification Mo ...
protein with two distinct functional domains. Site-directed
mutagenesis Mutagenesis () is a process by which the genetic information of an organism is changed by the production of a mutation. It may occur spontaneously in nature, or as a result of exposure to mutagens. It can also be achieved experimentally using lab ...
experiments support the
proposition In logic and linguistics, a proposition is the meaning of a declarative sentence. In philosophy, " meaning" is understood to be a non-linguistic entity which is shared by all sentences with the same meaning. Equivalently, a proposition is the no ...
that this protein displays a structural and functional similarity to the Klenow fragment of the ''Escherichia coli'' Polymerase I enzyme; 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 Helicases are a class of enzymes thought to be vital to all organisms. Their main function is to unpack an organism's genetic material. Helicases are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separatin ...
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.
Deoxyribonucleoside A deoxyribonucleotide is a nucleotide that contains deoxyribose. They are the monomeric units of the informational biopolymer, deoxyribonucleic acid ( DNA). Each deoxyribonucleotide comprises three parts: a deoxyribose sugar (monosaccharide), a nit ...
triphosphate cleavage that occurs as part of the polymerisation process probably supplies the energy required for this unwinding mechanism. 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 Directionality, in molecular biology and biochemistry, is the end-to-end chemical orientation of a single strand of nucleic acid. In a single strand of DNA or RNA, the chemical convention of naming carbon atoms in the nucleotide pentose-sugar-r ...
of the newly synthesised strand. 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 The wild type (WT) is the phenotype of the typical form of a species as it occurs in nature. Originally, the wild type was conceptualized as a product of the standard "normal" allele at a locus, in contrast to that produced by a non-standard, "m ...
enzyme.


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. *High processivity. *Proofreading activity. It is believed to be 1 or 2 orders of magnitude less error prone than Taq polymerase. *Generates large fragments, over 10kb. *Produces more DNA than PCR-based methods, by about an order of magnitude. *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.


References


Further reading

* * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *{{cite journal , vauthors=de Vega M, Lazaro JM, Salas M, Blanco L , title=Primer-terminus stabilization at the 3'-5' exonuclease active site of phi29 DNA polymerase. Involvement of two amino acid residues highly conserved in proofreading DNA polymerases. , journal=EMBO J , volume=15 , issue=5 , pages=1182–92 , year=1996 , pmid=8605889 , pmc=450017, doi=10.1002/j.1460-2075.1996.tb00457.x DNA replication Podoviridae