TempliPhi, phi29 DNA polymerase based rolling circle amplification of templates for DNA sequencing.


We have developed a novel, isothermal DNA amplification strategy that employs phi29 DNA polymerase and rolling circle amplification to generate high-quality templates for DNA sequencing reactions. The TempliPhi DNA amplification kits take advantage of the fact that cloned DNA is typically obtained in circular vectors, which are readily replicated in vitro using phi29 DNA polymerase by a rolling circle mechanism. This single subunit, proofreading DNA polymerase has excellent processivity and strand displacement properties for generation of multiple, tandem double-stranded copies of the circular DNA, generating as much as 10(7)-fold amplification. Large amounts of product (1-3 microg) can be obtained in as little as 4 hours. Input DNA can be as little as 0.01 ng of purified plasmid DNA, a single bacterial colony, or a 1 microL of a saturated overnight culture. Additionally, the presence of an associated proof reading function within the phi29 DNA polymerase ensures high-fidelity amplification. Once completed, the product DNA can be used directly in sequencing reactions. Additionally, the properties of phi29 DNA polymerase and its use in applications such as amplification ofhuman genomic DNA for genotyping studies is discussed.



Exonuclease Activity, Source / Purification, Fidelity, Nucleotide Incorporation, Kinetic Parameters


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Polymerase Reference Property Result Context
Phi29 Nelson JR2002 3-5' Exonuclease (proofreading) Yes
Phi29 Nelson JR2002 Cloned or native Native organism
Phi29 Nelson JR2002 Tagged No
Phi29 Nelson JR2002 Overall Error Rate 3E-06 errors/bp
Phi29 Nelson JR2002 Full length or truncated Full length
Phi29 Nelson JR2002 Maximum Product Length 800kb
Phi29 Nelson JR2002 Processivity 70kb
Phi29 Nelson JR2002 Vmax 50 /second Reaction: Nucleotide incorporation; Substrate: dNTPs

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