Use of transposon-promoted deletions in DNA sequence analysis.
The usefulness of the dideoxy method for DNA sequencing can be greatly extended by the use of transposon-generated deletions. These deletions have the unique property of extending from a fixed nucleotide at the transposon terminus to various sites outside it. A plasmid (pAA3.7) carrying Tn9, which allows positive selection of such deletions as galactose-resistant colonies of Escherichia coli, is described. A cloned gene can thus be subdivided into a series of overlapping sequences, all of which are fused to a common sequence at the transposon terminus. Restriction fragments carrying the segments fused by deletions are cloned in M13, and sequenced using a primer complementary to the Tn9 terminus. Complete nucleotide sequence of the gene is assembled from sequence overlaps found in deletions with end-points approximately 350 base-pairs apart. The method is rapid, requires minimal in vitro manipulation, and is free from redundant information normally produced in shotgun sequencing.
D001483:Base Sequence; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics*; D005690:Galactose / Q000235:genetics; D009154:Mutation; D010957:Plasmids
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D005690:Galactose