Nucleic Acids Research, Vol 27, Issue 19 3859-3865, Copyright © 1999 by Oxford University Press
TJ Griffin 4th, L Parsons, AE Leschziner, J DeVost, KM Derbyshire and ND Grindley
We have explored the potential of the Tn 552 in vitro transposition
reaction as a genetic tool. The reaction is simple (requiring a single
protein component), robust and efficient, readily producing insertions into
several percent of target DNA. Most importantly, Tn 552 insertions in vitro
appear to be essentially random. Extensive analyses indicate that the
transposon exhibits no significant regional or sequence specificity for
target DNA and leaves no discernible 'cold' spots devoid of insertions. The
utility of the in vitro reaction for DNA sequencing was demonstrated with a
cosmid containing the Mycobacterium smegmatis recBCD gene cluster. The
nucleotide sequence of the entire operon was determined using 71
independent Tn 552 insertions, which generated over 13.5 kb of unique
sequence and simultaneously provided a comprehensive collection of
insertion mutants. The relatively short ends of Tn 552 make construction of
novel transposons a simple process and we describe several useful
derivatives. The data presented suggest that Tn 552 transposition is a
valuable addition to the arsenal of tools available for molecular biology
and genomics.
ARTICLES
In vitro transposition of Tn552: a tool for DNA sequencing and mutagenesis
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
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