A SIMPLE METHOD FOR RAPID ISOLATION OF MICROSATELLITES FROM YEAST ARTIFICIAL CHROMOSOMES

Microsatellites are widely recognised as providing a rich source of po lymorphic markers for genetic mapping. Consequently, highly polymorphi c CA repeats tightly linked to a disease locus are invaluable tools in linkage studies. We have developed an efficient technique for cloning microsatellite repeats from a region of interest contained within a y east artificial chromosome (YAC). The YAC material is digested with a frequent cutting restriction endonuclease and ligated to polymerase ch ain reaction (PCR) amplifiable catch-linkers. A 5' biotinylated (CA)(1 1) oligonucleotide is then used to select fragments containing a compl ementary repeat by binding to streptavidin-coated magnetic beads. The catch-linkers enable these fragments to be PCR amplified, cloned and s equenced. Primers are then designed to amplify the repeat locus and to confirm its genomic localization and heterozygosity. We have successf ully used this technique to clone a new (CA)(18) microsatellite from a 360-kb YAC. The YAC contains the CYBB locus in Xp21.1 and is thought to contain at least part of the RP3 gene responsible for X-linked reti nitis pigmentosa. This new CA repeat is highly polymorphic with nine a lleles identified so far and a heterozygosity of 0.75.