LARGE DNA FRAGMENT CLONING IN BACTERIA BEYOND BACs AND PACs

Department of Soil and Crop Sciences and Crop Biotechnology Center, Texas A&M University, College Station, TX 77843-2123 USA

Large DNA fragment cloning are crucial to modern genomics research. Development of BAC and PAC systems for cloning of very large eukaryotic DNA fragments in bacteria has greatly contributed to genomics research. Dozens of large-insert BAC and PAC libraries have been developed for plants, animals and humans (http://hbz.tamu.edu). However, several theoretical questions of large DNA fragment cloning need to be answered which are extremely important for current genomics research. First, all currently used large DNA fragment cloning systems, YAC, BAC and PAC, are based on artificial chromosome systems, which seems that the construction of artificial chromosomes is essential for large DNA fragment cloning. The question has been raised if non-artificial chromosome systems, such as conventional plasmids and cosmids, can be used for large DNA fragment cloning. Second, studies have demonstrated that BACs and PACs are stable in their bacterial hosts and suggested that the stability of the BACs and PACs is due to their unique copy per cell. This concept has been widely accepted, however, further studies are needed to answer this question. Third, all existing BAC and PAC libraries have average insert sizes of ~150 kb, among which the largest BAC or PAC in insert size observed is ~350 kb. The question is why no BAC or PAC with an insert size >500 kb has been observed as with YACs. Is this due to the limited capacity of the BAC and PAC host, E.coli , in the stable maintenance of foreign DNA fragments or the limitation of the currently used cloning techniques? In this presentation, we will show that conventional plasmid-based vectors allow cloning and stable maintenance of DNA fragments >300 kb in E.coli. The bacterium E.coli strain DH10B is able to stably maintain at least 1200 kb of foreign DNA fragments in the form of plasmids in a single cell. We will also show that the stability of large-insert clones does not seem contingent on the single copy status of the clones. These results urge a re-evaluation of the dogma for large DNA fragment cloning and suggest a new conceptual and theoretical basis for large DNA fragment cloning and plant and animal engineering beyond current BACs and PACs. According to this discovery, we are developing technologies for constructing bacterial clone libraries with average insert sizes of 200 - 500 kb.