TY - JOUR
T1 - Isothermal DNA amplification using the T4 replisome
T2 - Circular nicking endonuclease-dependent amplification and primase-based whole-genome amplification
AU - Schaerli, Yolanda
AU - Stein, Viktor
AU - Spiering, Michelle M.
AU - Benkovic, Stephen J.
AU - Abell, Chris
AU - Hollfelder, Florian
N1 - Funding Information:
Ernst Schering Foundation and the Cambridge Overseas Trust and Trinity Hall, Cambridge (to Y.S.); Trinity College, Cambridge, and the Biotechnology and Biological Sciences Research Council (BBSRC) (to V.S.); a Human Frontier Science Program grant and US National Institutes of Health grant GM013306 (to S.J.B.); EU NEST MiFem and an RCUK Basic Technology Grant. F.H. is an ERC Starting Investigator. Funding for open access charge: RCUK Basic Technology grant.
PY - 2010/12
Y1 - 2010/12
N2 - 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.
AB - 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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U2 - 10.1093/nar/gkq795
DO - 10.1093/nar/gkq795
M3 - Article
C2 - 20921065
AN - SCOPUS:78650433287
SN - 0305-1048
VL - 38
SP - e201
JO - Nucleic acids research
JF - Nucleic acids research
IS - 22
ER -