An efficient DNA sequencing strategy based on the bacteriophage Mu in vitro DNA transposition reaction

highly efficient DNA sequencing strategy was developed on the basis of the bacteriophage Mu in vitro DNA transposition reaction. In the reaction, an a rtificial transposon with a chloramphenicol acetyltransferase [cat) gene as a selectable marker integrated into the target plasmid DNA containing a 10 .3-kb mouse genomic insert to be sequenced. Bacterial clones carrying plasm ids with the transposon insertions in different positions were produced by transforming transposition reaction products into Escherichia coli cells th at were then selected on appropriate selection plates. Plasmids from indivi dual clones were isolated and used as templates For DNA sequencing, each wi th two primers specific for the transposon sequence but reading the sequenc e into opposite directions, thus creating a minicontig. By combining the in formation from overlapping minicontigs, the sequence of the entire 10,288-b p region of mouse genome including six exons of mouse Kcc2 gene was obtaine d. The results indicated that the described methodology is extremely well s uited For DNA sequencing projects in which considerable sequence informatio n is on demand. In addition, massive DNA sequencing projects, including tho se of full genomes, are expected to benefit substantially from the Mu strat egy.