Excision and amplification of large genomic segments in natural hosts

Abstract

In vivo excision and amplification of pre-determined, large genomic segments, directly from the genome of a natural host, provides an alternative to conventional cloning in foreign vectors. The prototype of our approach was the excisional replication of some viruses such as the lambda lysogen. A similar machinery was used to excise and amplify large genomic segments from the original organisms. This approach involved the insertion of two recognition sequences, loxPs, for the site-specific recombinase, Cre, into the genome at pre-determined sites, 50-100 kb apart. The integration of these sequences, together with a conditional replication origin (ori), was targeted by homologous recombination. The strain carrying the insertions was stably maintained until, upon induction of specifically engineered genes, the host cell expressed the site-specific recombinase and an ori-specific replication protein. The recombinase then excised and circularized the genomic segment flanked by the two loxP sites. This excised and circularized DNA, which contained the ori sequence, was amplified with the aid of the replication protein and could be isolated as large plasmids. We have devised three in vivo procedures for excising large segments of Escherichia coli, Saccharomyces cerevisiae and human genome using bacteriophage P1 Cre/loxP system. For amplification of the excised circular DNAs, E. coli R6K plasmid-derived π/γ-ori replication system, yeast 2 ㎛ plasmid-derived Flp/FRT/2 ㎛-ori system, and Epstein-Barr virus-derived EBNA1/oriP or Simian Virus 40-derived large T antigen/SV40-ori replication machinery were used, respectively. Applying these procedures, the 50-kb and 100-kb DNA segments of E. coli, S. cerevisiae and human genomes were successfully excised and in vivo amplified by the amplification functions. Such excised and amplified genomic DNA segments can be used for the sequencing the genome and functional analysis of any genes of each organism.