Rapid Amplification of Genomic DNA Sequences Tagged by Insertional Mutagenesis

Current and recent efforts to determine the genomic DNA sequence for numerous organisms (e.g., Saccharomyces cerevisiae, Candida albicans, Neurospora crassa, Arabidopsis thaliana, Zea mays, Caenorhabditis elegans, Mus musculus, Homo sapiens, Schizosaccharomyces pombe, Danio rerio, Drosophila melanogaster, Oryza sativa , and various archaea, and eubacteria) have revealed novel genes with unknown functions, and transposon mutagenesis provides a powerful method for assigning functions to these genes (e.g., 1 ,2 -5 ). In addition, restriction enzyme-mediated integration (REMI) has been used widely for mutagenesis (e.g., 6 ,7 , reviewed in 8 ). For both of these approaches, a unique DNA sequence is inserted by nonhomologous integration into genomic DNA to generate a disruption mutation that is physically tagged. Genetic analysis of the resulting mutants yields those with defects in the function of interest. Because each mutation is physically marked by a unique DNA sequence, it is possible to use this tag to identify each disrupted gene.