A directed DNA sequencing strategy based upon Tn3 transposon mutagenesis: application to the ADE1 locus on Saccharomyces cerevisiae chromosome I.

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RESUMO

We have developed a directed DNA sequencing strategy based upon the Escherichia coli transposon Tn3. This transposon displays little sequence specificity for transposition and is thus well suited to this task. Both mini-Tn3 transposons and sequencing vectors bearing the phage f1 single stranded origin of replication have been constructed. Upon mutagenesis of a target sequence, a population is produced in which each clone has two f1 origins of replication, one of which is at a variable position depending upon the transposon insertion site. When helper phage is added to the mutagenised population, the two f1 origins present on each clone are nicked, dividing the packaged strand into two segments, each of which is packaged into a separate phage particle. One of these segments contains no resistance markers and is lost, whilst the other is recovered as a deleted clone with a single chimeric f1 origin. A unidirectionally, variably-deleted set of sequencing clones is produced, and appropriately sized clones are sequenced using a primer complimentary to the transposon end. In addition to being inexpensive, the method does not require the same degree of technical expertise needed for many in vitro, enzymatically based methods. The strategy has been used to determine 2.6 kilobases of nucleotide sequence in the Saccharomyces cerevisiae ADE 1 locus.

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