Isothermal DNA amplification using the T4 replisome

Circular nicking endonuclease-dependent amplification and primase-based whole-genome amplification

Yolanda Schaerli, Viktor Stein, Michelle Marie Spiering, Stephen Benkovic, Chris Abell, Florian Hollfelder

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

In vitro reconstitution of the bacteriophage T4 replication machinery provides a novel system for fast and processive isothermal DNA amplification. We have characterized this system in two formats: (i) in circular nicking endonuclease-dependent amplification (cNDA), the T4 replisome is supplemented with a nicking endonuclease (Nb.BbvCI) and a reverse primer to generate a well-defined uniform double-stranded linear product and to achieve up to 1100-fold linear amplification of a plasmid in 1h. (ii) The T4 replisome with its primase (gp61) can also support priming and exponential amplification of genomic DNA in primase-based whole-genome amplification (T4 pWGA). Low amplification biases between 4.8 and 9.8 among eight loci for 0.3-10ng template DNA suggest that this method is indeed suitable for uniform whole-genome amplification. Finally, the utility of the T4 replisome for isothermal DNA amplification is demonstrated in various applications, including incorporation of functional tags for DNA labeling and immobilization; template generation for in vitro transcription/translation and sequencing; and colony screening and DNA quantification.

Original languageEnglish (US)
JournalNucleic acids research
Volume38
Issue number22
DOIs
StatePublished - Dec 1 2010

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DNA Primase
Endonucleases
Genome
DNA
Bacteriophage T4
Immobilization
Plasmids
In Vitro Techniques

All Science Journal Classification (ASJC) codes

  • Genetics

Cite this

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title = "Isothermal DNA amplification using the T4 replisome: Circular nicking endonuclease-dependent amplification and primase-based whole-genome amplification",
abstract = "In vitro reconstitution of the bacteriophage T4 replication machinery provides a novel system for fast and processive isothermal DNA amplification. We have characterized this system in two formats: (i) in circular nicking endonuclease-dependent amplification (cNDA), the T4 replisome is supplemented with a nicking endonuclease (Nb.BbvCI) and a reverse primer to generate a well-defined uniform double-stranded linear product and to achieve up to 1100-fold linear amplification of a plasmid in 1h. (ii) The T4 replisome with its primase (gp61) can also support priming and exponential amplification of genomic DNA in primase-based whole-genome amplification (T4 pWGA). Low amplification biases between 4.8 and 9.8 among eight loci for 0.3-10ng template DNA suggest that this method is indeed suitable for uniform whole-genome amplification. Finally, the utility of the T4 replisome for isothermal DNA amplification is demonstrated in various applications, including incorporation of functional tags for DNA labeling and immobilization; template generation for in vitro transcription/translation and sequencing; and colony screening and DNA quantification.",
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Isothermal DNA amplification using the T4 replisome : Circular nicking endonuclease-dependent amplification and primase-based whole-genome amplification. / Schaerli, Yolanda; Stein, Viktor; Spiering, Michelle Marie; Benkovic, Stephen; Abell, Chris; Hollfelder, Florian.

In: Nucleic acids research, Vol. 38, No. 22, 01.12.2010.

Research output: Contribution to journalArticle

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T2 - Circular nicking endonuclease-dependent amplification and primase-based whole-genome amplification

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AU - Benkovic, Stephen

AU - Abell, Chris

AU - Hollfelder, Florian

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