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

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Version: Final published version
Serval ID
serval:BIB_4495846F6EC3
Type
Article: article from journal or magazin.
Collection
Publications
Title
Isothermal DNA amplification using the T4 replisome: circular nicking endonuclease-dependent amplification and primase-based whole-genome amplification.
Journal
Nucleic Acids Research
Author(s)
Schaerli Y., Stein V., Spiering M.M., Benkovic S.J., Abell C., Hollfelder F.
ISSN
1362-4962 (Electronic)
ISSN-L
0305-1048
Publication state
Published
Issued date
2010
Peer-reviewed
Oui
Volume
38
Number
22
Pages
e201
Language
english
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 1 h. (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 0.3-10 ng 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.

Keywords
Bacteriophage T4/enzymology, Bacteriophage T4/genetics, DNA Primase/metabolism, DNA-Directed DNA Polymerase/metabolism, Endodeoxyribonucleases/metabolism, Genome, Human, Humans, Multienzyme Complexes/metabolism, Nucleic Acid Amplification Techniques/methods, Plasmids/genetics, Sequence Analysis, DNA, Templates, Genetic, Transcription, Genetic, Translocation, Genetic
Pubmed
Web of science
Open Access
Yes
Create date
02/02/2017 8:11
Last modification date
20/08/2019 13:49
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