Rapid Cloning of Novel Genes and Promoters for Functional Analyses in Transgenic Cells PEER M. SCHENK 1,2,* , CLAUDIA E. VICKERS 2,3 AND JOHN M. MANNERS 1,3 1 Cooperative Research Centre for Tropical Plant Protection, Level 5 John Hines Building, 2 Department of Botany, School of Life Sciences, The University of Queensland, St. Lucia, QLD 4072, 3 CSIRO Plant Industry, 306 Carmody Rd, St Lucia QLD 4067, Australia 151 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 sys- tems that involve initial cloning into a spe- cialized 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 function- al 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. Key Words: amplification, functional genomics, gene mining, library construction, rapid cloning, transgenic expression INTRODUCTION The recent availability of large data sets of sequence information (e.g. large-scale EST and whole genome sequencing projects) is ____________________ *Corresponding author: E-mail: p.schenk@uq.edu.au; Fax: +61-7-33654771; Phone: +61-7-33658817 Transgenics, Vol. 4, pp. 151-156 © 2004 Old City Publishing, Inc. Reprints available directly from the publisher Published by license under the OCP Science imprint, Photocopying permitted by license only a member of the Old City Publishing Group.