MAPPING EXPRESSED SEQUENCE TAG SITES ON YEAST ARTIFICIAL CHROMOSOME CLONES OF ARABIDOPSIS-THALIANA DNA

We describe a method for efficient parallel mapping of expressed seque nce tag (EST) sites onto yeast artificial chromosome (YAC) clones. The strategy involves an initial YAC clone pooling scheme that minimizes the number of required PCR amplifications. This is followed by paralle l analysis of PCR amplicons of EST sequences. Using this method, we ha ve screened 600 EST sites in combinatorial pools of 3449 YAC clones th at contain Arabidopsis thaliana DNA inserts. The presence of these gen es on YACs was detected by amplifying EST sequences with PCR and analy zing the reaction products by agarose gel electrophoresis. Of the 600 ESTs, 271 were found to map to individual YACs. Software tools are pre sented that allow for the automated analysis of this electrophoresis d ata. Suggestions for the scale-up of this method to map large genomes are discussed.