Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

Nucleic Acids Research logoLink to Nucleic Acids Research
. 1999 Oct 1;27(19):3859–3865. doi: 10.1093/nar/27.19.3859

In vitro transposition of Tn552: a tool for DNA sequencing and mutagenesis.

T J Griffin 4th 1, L Parsons 1, A E Leschziner 1, J DeVost 1, K M Derbyshire 1, N D Grindley 1
PMCID: PMC148649  PMID: 10481025

Abstract

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.

Full Text

The Full Text of this article is available as a PDF (290.8 KB).


Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES