ISOLATION OF MURINE TELOMERE-PROXIMAL SEQUENCES BY AFFINITY CAPTURE AND PCR

We describe a method of selectively enriching for murine telomere-prox imal sequences using affinity capture followed by PCR amplification. T he telomeric fragments were selected from NotI-digested and lambda exo nuclease-resected mouse genomic DNA by annealing to a biotinylated rib oprobe containing multiple copies of the telomere repeat (TTAGGG)(n). The resultant DNA-RNA hybrids were selectively retained on a matrix wi th covalently bound avidin. The captured DNA was then specifically rel eased by ribonuclease action, and PCR amplification was performed usin g mouse repeat primers. The PCR products were cloned and used to scree n a mouse genomic cosmid library, and the resultant cosmid clones were analyzed by fluorescence in situ hybridization. Ten of 70 clones anal yzed gave telomere-proximal hybridization signals, indicating an at le ast 500-fold enrichment for telomere-proximal sequences. (C) 1995 Acad emic Press, Inc.