Probe mapping to facilitate transposon-based DNA sequencing.

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RESUMO

A promising strategy for DNA sequencing exploits transposons to provide mobile sites for the binding of sequencing primers. For such a strategy to be maximally efficient, the location and orientation of the transposon must be readily determined and the insertion sites should be randomly distributed. We demonstrate an efficient probe-based method for the localization and orientation of transposon-borne primer sites, which is adaptable to large-scale sequencing strategies. This approach requires no prior restriction enzyme mapping or knowledge of the cloned sequence and eliminates the inefficiency inherent in totally random sequencing methods. To test the efficiency of probe mapping, 49 insertions of the transposon gamma delta (Tn1000) in a cloned fragment of Drosophila melanogaster DNA were mapped and oriented. In addition, oligonucleotide primers specific for unique subterminal gamma delta segments were used to prime dideoxynucleotide double-stranded sequencing. These data provided an opportunity to rigorously examine gamma delta insertion sites. The insertions were quite randomly distributed, even though the target DNA fragment had both A + T-rich and G + C-rich regions; in G + C-rich DNA, the insertions were found in A + T-rich "valleys." These data demonstrate that gamma delta is an excellent choice for supplying mobile primer binding sites to cloned DNA and that transposon-based probe mapping permits the sequences of large cloned segments to be determined without any subcloning.

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