Transposon-facilitated sequencing of DNAs cloned in plasmids.

Publisher Summary Transposon insertion sites can be readily mapped, so redundant sequencing can be avoided, and gap closure, if necessary, is simplified. Problems associated with sequencing repetitive DNA are minimized. Bacterial transposons show great promise as tools for both small and large-scale sequencing efforts, by virtue of their ability to move and carry useful specific primer-binding sites to many locations in cloned DNA targets. Many new methods should interface well with transposon-based template generating methods. Among them are (1) direct polymerase chain reaction (PCR) for amplifying DNA segments, using a transposon to provide one primer-binding site,(2) crossover PCR to amplify DNA segments delimited by independent transposon insertions in different molecules, (3) multiplex sequencing using transposon-carried multiplex tags, (4) linear amplification (cycle) sequencing, a one-ended PCR reaction, using a single transposon primer in the presence of chain-terminating nucleotides, (5) primer walking from transposon anchor sites using preformed oligomer libraries, and (6) exometh sequencing using a transposon to provide appropriate restriction sites.