Use of Dpn I Restriction Enzyme to Assess Newly Replicated Gene Copies in Amplifiable Vector Systems

Abstract

Bacterially propagated plasmid DNA can be transfected into established eukaryotic cell lines or primary cell cultures by a variety of techniques, such as electroporation (see Chapter 5, this vol) (1), scrape-loading (2), and DEAE dextran (see Chapter 3) or calcium phosphate mediated gene transfer (see Chapter 2) (3–5). At least some of the DNA introduced into the cells enters into the nucleus, where it is thought to be assembled into chromatin (6), and is maintained extrachromosomally for at least 48 h. During this time, the cellular chromosomal DNA may have undergone one or more rounds of DNA replication. However, the extrachromosomal transfected DNA will not replicate unless the DNA sequences contained in the plasmid include a DNA origin of replication recognized by the host cell. Origin sequences have so far proved difficult to identify in eukaryotic chromosomes. In contrast, viral genomes, such as SV40 and polyoma, have well-characterized origins of replication (7), which, when included on DNA