Isothermal DNA amplification using the T4 replisome: Circular nicking endonuclease-dependent amplification and primase-based whole-genome amplification

Yolanda Schaerli, Viktor Stein, Michelle M. Spiering, Stephen J. Benkovic, Chris Abell, Florian Hollfelder

Research output: Contribution to journalArticle

19 Scopus citations

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)
Pages (from-to)e201
JournalNucleic acids research
Volume38
Issue number22
DOIs
StatePublished - Dec 2010

All Science Journal Classification (ASJC) codes

  • Genetics

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