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Nucleic Acids Research, 1992, Vol. 20, No. 14 3665-3669
© 1992

METHODS

A new strategy useful rapid identification of microsatellites from DNA libraries with large size inserts

Bruno Baron, Christophe Poirier, Dominique Simon-Chazottes, Cécile Barnier and Jean-Louis Guénet

Unité de Génétique des Mammiféres de l'Institut Pasteur 25 rue de Docteur Roux, 75724 Paris Cedex 15, France

Received March 30, 1992. Revised June 26, 1992. Accepted June 26, 1992.

Microsatellites are new powerful polymorphic markers used for gene mapping. Their characterization requires that all the sequence surrounding the repeat be known in order to be able to design primers for PCR amplification. However, when using DNA libraries with large cloned inserts, this sequence characterization Is not immediately practicable. In this paper, we describe a new strategy, based both on the use of a microsatellite specific probing and on the creation of nested deleted clones with the Exonuclease III, in order to position microsatellites in a range allowing direct sequencing. This method was applied to the screening of a mouse chromosome 19 DNA specific library. In this way, thirteen clones were identified by specific probing and seven were submitted to the nested deletion strategy. Five of them presented microsatellite sequences in specific deleted subclones which were selected and sequenced. Primers were designed for each of them and polymorphism between the genomes of several inbred strain of mouse have been determined. These microsatellites were mapped, three of them to chromosome 19 and two to chromosome 11.


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