Amplification of closed circular DNA in vitro

The polymerase chain reaction is a powerful technique used to amplify nucleic acids in vitro. The reaction produces linear products, and as of yet, closed circular products have not been possible. Since the replicatively competent form of many DNA molecules is the closed circular form, it would be adventitious to amplify closed circular DNA as closed circular molecules. Until now, these molecules could only be amplified in vivo in appropriate host cells. Here, we describe an in vito procedure, ligation-during-amplification (LDA), for selective amplification of closed circular DNA using sequence-specific primers. LDA is useful for site-directed mutagenesis, mutation detection, DNA modification, DNA library screening and circular DNA production.