Rapid Cloning of Novel Genes and Promoters for Functional Analyses in Transgenic Cells
Peer M. Schenk, Claudial E. Vickers and John M. Manners
The availability of sequence information for thousands of genes for many organisms is currently unmatched by functional studies. A cost-effective and high-throughput cloning system for PCR products was therefore adopted to enable the rapid assessment of coding and promoter sequences in functional assays in transgenic cells. Unlike other systems that involve initial cloning into a specialized PCR fragment cloning vector, this method describes a rapid and cost-effective procedure for the amplification of a DNA fragment by PCR, its phosphorylation and its direct insertion into the vector of choice. Restriction enzymes are only required once for the preparation of the recipient vector, which is blunt-ended and dephosphorylated. No special primer designs (e.g. restriction enzyme sites or flanking homologous sequences) or subcloning steps are required. The turn-around time from source organism genomic DNA to new recombinant DNA is 24 hrs. It is particularly suitable for functional genomics projects or the generation of libraries from PCR products where a large number of fragments need to be cloned into the same vector. We have used this method to rapidly clone 72 full-length genes (ranging from 0.8 to 6.4 kb) and putative promoters (2 kb each) from Arabidopsis thaliana into plant cell expression cassettes for subsequent direct functional analyses in transgenic cells.