ISOLATION OF YAC INSERT SEQUENCES BY REPRESENTATIONAL DIFFERENCE ANALYSIS

We present a method for the isolation of YAC insert sequences by repre sentational difference analysis (RDA). To achieve maximal representati on of the sequences, the amplicons were generated from a Mbol digestio n product. RDA was performed using a 970 kb insert YAC clone. After tw o rounds of re-association and selective amplification 92% of the diff erence product represented sequences derived from the YAC insert. Twen ty insert-specific sequence-tagged sites were readily defined. The dif ference product was also successfully used to isolate microsatellite m arkers, to identify clones from a human PAC library and as a chromosom e painting probe in fluorescence in situ hybridization.