Abstracts: 



Sequencing 



Technologies 



Transposon-Facilitated DNA Sequencing 



Douglas E. Berg, Clara M. Berg, and Henry Huang 



Department ot Microbiology and Immunology. Washington University, 



St. Louis. MO 631 10 



(314)362-2772 



Two types of derivatives of transposon Tn5 will be constructed to facilitate the 

 sequencing of cloned DN As. One type is designed for in vivo insertion at many sites in 

 DNAs cloned in lambda phage and in cosmids and other plasmid vectors, and for 

 sequencing in both directions from each site of insertion. The other type will be 

 embedded in cosmids and used to generate nested deletions with one variable end point 

 in the cloned DNA and one end point fixed at a transposon end; the set of nested 

 deletions will similarly permit sequencing of the entire stretch of cloned DNA without 

 need for subcloning of random fragments. The Tn5 element for insertion into lambda 

 and cosmids will contain a supF (suppressor tRNA) gene as a selectable marker. Its 

 transposition to lambda will be selected by plaque formation, while its transposition to 

 plasmids will be selected by suppression of a chromosomal amber mutation. Constructs 

 for making nested deletions by intramolecular transposition will contain a Tn5 

 transposase gene, whose expression is turned on by IPTG. so that deletions can be made 

 at will. The construct will also contain a conditionally lethal gene for selection of the 

 transposition-induced deletions. Deletion-generating Tn5 derivatives will be constructed 

 as complete cosmid vectors for the construction of new recombinant DNA libraries and 

 as cassettes for insertion into cosmids that already contain cloned DNAs. Both types of 

 Tn5 derivatives will be adapted for multiplex sequencing. 



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