Schaerli, Yolanda ; Stein, Viktor ; Spiering, Michelle M. ; Benkovic, Stephen J. ; Abell, Chris ; Hollfelder, Florian (2010)
Isothermal DNA amplification using the T4 replisome: circular nicking endonuclease-dependent amplification and primase-based whole-genome amplification.
In: Nucleic acids research, 38 (22)
Artikel, Bibliographie
Kurzbeschreibung (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.
| Typ des Eintrags: | Artikel |
|---|---|
| Erschienen: | 2010 |
| Autor(en): | Schaerli, Yolanda ; Stein, Viktor ; Spiering, Michelle M. ; Benkovic, Stephen J. ; Abell, Chris ; Hollfelder, Florian |
| Art des Eintrags: | Bibliographie |
| Titel: | Isothermal DNA amplification using the T4 replisome: circular nicking endonuclease-dependent amplification and primase-based whole-genome amplification. |
| Sprache: | Englisch |
| Publikationsjahr: | 2010 |
| Titel der Zeitschrift, Zeitung oder Schriftenreihe: | Nucleic acids research |
| Jahrgang/Volume einer Zeitschrift: | 38 |
| (Heft-)Nummer: | 22 |
| Kurzbeschreibung (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. |
| Fachbereich(e)/-gebiet(e): | 10 Fachbereich Biologie 10 Fachbereich Biologie > Protein Engineering of Ion Conducting Nanopores |
| Hinterlegungsdatum: | 14 Nov 2016 11:41 |
| Letzte Änderung: | 14 Nov 2016 11:41 |
| PPN: | |
| Export: | |
| Suche nach Titel in: | TUfind oder in Google |
 |
Frage zum Eintrag |
Optionen (nur für Redakteure)
 |
Redaktionelle Details anzeigen |
Drucken
Impressum