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Rapid sequencing of cloned DNA using a transposon for bidirectional priming: sequence of the Escherichia coli K-12 avtA gene.

Authors
  • L Liu
  • W Whalen
  • A Das
  • C M Berg
Publication Date
Nov 25, 1987
Source
PMC
Disciplines
  • Mathematics
License
Unknown

Abstract

A new approach to determining the sequence of cloned DNA is described. Unique regions near each end of the transposable element gamma-delta provide a pair of "portable" primer-specific sites for bidirectional sequencing by the dideoxy chain termination method. A set of gamma-delta insertions positioned about 200 bp apart over the entire cloned DNA allowed us to determine the sequence of both strands in a single parental plasmid without subcloning. The avtA (alanine-valine transaminase) gene of E. coli K-12 was sequenced by this approach. Surprisingly, gamma-delta insertions downstream of the coding region were found to significantly reduce avtA expression. We suggest that these nondisruptive insertions probably change the DNA topology and thereby alter gene expression.

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