The use of exonuclease III deletions in automated DNA sequencing

Abstract

We have optimized the use of Exonuclease III deletions as a sequencing strategy for use with Applied Biosystems 373 DNA sequencers. We describe how we carry out the procedure using a gel purification step to more rigidly control the size of the deletions and to eliminate improperly cut plasmid DNA from subsequent sequencing. We also present procedures for single-stranded phagemid rescue and double-stranded plasmid isolation. These methods, coupled with the new cycle-sequencing reactions, allow virtually failure-free Exo III deletions and sequencing to be performed. Also described are our ongoing efforts to develop a more efficient method for carrying out Exo III deletions. These include the use of a specialized preparative electrophoresis device to effect the size separation of deleted fragments and the use of a robotic workstation to carry out the Exo III deletion steps. We use Exo III deletions for sequencing cDNA clones and small fragments of chromosomal DNA. We are also using this method to fill gaps remaining between contigs in shotgun sequencing projects carried out on cosmids and to confirm the sequence of repetitive regions of cosmids.