Sequencing Methods

The methodology used is a classical shotgun sequencing strategy. A random library of the BAC/cosmid is obtained by sonication. The E.coli colonies/clones from the library are grown in a liquid culture in 96-well microplates for 22 hours at 37 °C. For each clone the DNA is then extracted and purified following a protocole set up at the Sanger Centre (Hinxton Hall, Cambridge, UK).

The sequencing reactions are done either with a "BigDye Terminator" kit from ABI (Perkin-Elmer) or with a "DYEnamic^TM ET Terminator" kit from Amersham-Pharmacia. The reactions are loaded on a 96-well acrylamide gel enabling the separation of DNA fragments labelled by fluorescence with a size difference as little as 1 nucleotide. The detection of the fragments is performed by the laser beam of an ABI377 DNA sequencer upgraded to 96 wells.

The independant sequences obtained for every single clone are aligned with the Phred-Phrap (Ewing-Gordon-Green, Washington) and GAP4 (Staden, Cambridge) assembly software running on a PC powered by Linux.