Construction of gene disruption mutants and analysis of the resultant phenotypes are an important strategy to study gene function. A simple and high-throughput method developed for microorganisms combines two different types of transposons, direct genomic DNA amplification and thermal asymmetric interlaced-PCR. The considerable utility of this approach is demonstrable in Corynebacterium glutamicum , where 18,000 transposon disruptants enabled the generation of an insertion library covering nearly 80% of the organism’s 2,990 ORFs.