Stable propagation of cosmid sized human DNA inserts in an F factor based vector

Abstract

Instability of complex mammalian genomic DNA inserts is commonplace in cosmid libraries constructed in conventional multicopy vectors. To develop a means to construct stable libraries, we have developed a low copy number cosmid vector based on the E.coli F factor replicon (Fosmid). We have tested relative stability of human DNA inserts in Fosmlds and in two conventional multicopy vectors (Lawrist 16 and Supercos) by comparing the frequency of changes In restriction patterns of the inserts after propagating randomly picked human genomic clones based on these vectors. We found that the clones based on Fosmid vector undergo detectable changes at a greatly reduced frequency. We also observed that sequences that undergo drastic rearrangements and deletions during propagation In a conventional vector were stably propagated when recloned as Fosmids. The results indicate that Fosmid system may be useful for constructing stable libraries from complex genomes.