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Isothermal DNA amplification using the T4 replisome: circular nicking endonuclease-dependent amplification and primase-based whole-genome amplification.
Nucleic Acids Research
Date de publication
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.
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
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