A new strategy useful for rapid identification of microsatellites from DNA libraries with large size inserts.
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.
Baron
B
B
Unité de Génétique des Mammifères, l'Institut Pasteur, Paris, France.
Poirier
C
C
Simon-Chazottes
D
D
Barnier
C
C
Guénet
J L
JL
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D015723:Gene Library; D005819:Genetic Markers; D005820:Genetic Testing / Q000379:methods*; D051379:Mice; D008815:Mice, Inbred Strains / Q000235:genetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*
D005819:Genetic Markers; D009838:Oligodeoxyribonucleotides