Probe mapping to facilitate transposon-based DNA sequencing.

Abstract

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 vy (TnlOOO) in a cloned fragment of Drosophila melanogaster DNA were mapped and oriented. In addition, oligonucleotide primers specific for unique subterminal y8 segments were used to prime dideoxynucleotide double-stranded sequencing. These data provided an opportunity to rigorously examine yO 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 yv 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.The current commitment to determining the sequence of the genomes of humans and other model organisms is stimulating technical advances in molecular genetics and giving new insights into genome structure, gene function, and evolution. Techniques for the rapid determination of nucleotide sequence have been developed (1, 2), and vectors such as cosmids (3) and bacteriophage P1 (4) permit large genomic segments to be cloned in Escherichia coli. Typically, large cloned DNAs are fragmented so that relatively short pieces can be brought into juxtaposition with fixed primer binding sites for sequencing, either by subcloning or by the generation of nested deletions (5). Even larger DNA segments may be sequenced directly by oligomer walking (6), by using a library of primers (7), or by the ExoMeth method (8).A fundamentally different sequencing approach that uses transposons to provide mobile sites for primer binding has several attractive features: subcloning and/or the isolation of nested deletions are not required, only two primers are needed, and large cloned fragments can be sequenced directly. Transposons have been successfully used to provide DNA sequencing primer sites in plasmids, phage A, and in chromosomal DNA (9)(10)(11)(12)(13)(14). However, even with transposons, sequence acquisition is either random and therefore highly repetitive, or it requires the time-consuming construction of a restriction map to locate the insertions. Furthermore, the usefulness of transposon-borne primer sites may be limited if the transposon inserts nonrandomly.We demonstrate here an effici...

Overlapping substrate specificities of benzaldehyde dehydrogenase (the xylC gene product) and 2-hydroxymuconic semialdehyde dehydrogenase (the xylG gene product) encoded by TOL plasmid pWW0 of Pseudomonas putida

“…DNA sequencing with double-stranded DNA was performed with the T7 DNA polymerase kit from Pharmacia (Uppsala, Sweden). Nucleotide sequencing of plasmid DNA around Tn1000 insertions was carried out with Tn1000-specific oligonucleotides as primers (22). The computer-assisted sequence analysis was done with the PC/GENE software package (IntelliGenetics, Mountain View, Calif.).…”

Transposon tools for recombinant DNA manipulation: characterization of transcriptional regulators from yeast, Xenopus, and mouse.

“…§1734 solely to indicate this fact. (4)(5)(6). Finally, TnlOOO has the major practical advantage of having a simple, one-step delivery system to produce a population of entirely transposed plasmids (9).…”

Transposon Tn5supF-based reverse genetic method for mutational analysis of Escherichia coli with DNAs cloned in lambda phage

“…TnS and related elements typically insert into dozens of sites per gene and also into certain sites preferentially (4,5,18). Consequently, mutagenesis to near saturation may sometimes benefit from combined use of two unrelated transposons, for example, a small supF-containing derivative of -y5, which also inserts into many sites with little specificity (3,9,29), along with TnSsupF. We thus anticipate that transposon-based reverse genetics with DNAs cloned in contraselectable vectors will make it feasible to carry out large-scale functional tests that will be important complements to E. coli genome sequencing efforts.…”