Format

Send to

Choose Destination
Proc Natl Acad Sci U S A. 2011 Nov 15;108(46):18655-60. doi: 10.1073/pnas.1114397108. Epub 2011 Nov 7.

Terminal protein-primed amplification of heterologous DNA with a minimal replication system based on phage Phi29.

Author information

1
Instituto de Biología Molecular Eladio Viñuela, Centro de Biología Molecular, Universidad Autónoma, Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.

Abstract

The DNA amplification performed by terminal protein-primed replication systems has not yet been developed for its general use to produce high amounts of DNA linked to terminal protein (TP). Here we present a method to amplify in vitro heterologous DNAs using the Φ29 DNA replication machinery and producing DNA with TP covalently attached to the 5' end. The amplification requires four Φ29 proteins, DNA polymerase, TP, single-stranded DNA binding protein and double-stranded DNA binding protein (p6). The DNA to be amplified is inserted between two sequences that are the Φ29 DNA replication origins, consisting of 191 and 194 bp from the left and right ends of the phage genome, respectively. The replication origins do not need to have TP covalently attached beforehand to be functional in amplification and they can be joined to the DNA to be amplified by cloning or ligation. The facts that two functional origins were required at the ends of a linear template DNA and that the kinetics of DNA synthesis was very similar to that obtained using the TP-containing Φ29 genome as template support the proposal that genuine amplification is taking place. Amplification factors of 30-fold have been obtained. Possible applications of DNAs produced by this method are discussed.

PMID:
22065756
PMCID:
PMC3219123
DOI:
10.1073/pnas.1114397108
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center