Terminal protein-primed amplification of heterologous DNA with a minimal replication system based on phage Φ29

Abstract

The DNA amplification performed by terminal protein-primed repli-cation systems has not yet been developed for its general use toproduce high amounts of DNA linked to terminal protein (TP). Herewe present a method to amplify in vitro heterologous DNAs usingthe Φ29 DNA replication machinery and producing DNA with TPcovalently attached to the 5′ end. The amplification requires fourΦ29 proteins, DNA polymerase, TP, single-stranded DNA bindingprotein and double-stranded DNA binding protein (p6). The DNAto be amplified is inserted between two sequences that are theΦ29 DNA replication origins, consisting of 191 and 194 bp fromthe left and right ends of the phage genome, respectively. Thereplication origins do not need to have TP covalently attachedbeforehand to be functional in amplification and they can be joinedto the DNA to be amplified by cloning or ligation. The facts thattwo functional origins were required at the ends of a linear tem-plate DNA and that the kinetics of DNA synthesis was very similarto that obtained using the TP-containing Φ29 genome as templatesupport the proposal that genuine amplification is taking place.Amplification factors of 30-fold have been obtained. Possibleapplications of DNAs produced by this method are discussed.