Isolation of murine telomere-proximal sequences by affinity capture and PCR.
We describe a method of selectively enriching for murine telomere-proximal sequences using affinity capture followed by PCR amplification. The telomeric fragments were selected from NotI-digested and lambda exonuclease-resected mouse genomic DNA by annealing to a biotinylated riboprobe containing multiple copies of the telomere repeat (TTAGGG)n. The resultant DNA-RNA hybrids were selectively retained on a matrix with covalently bound avidin. The captured DNA was then specifically released by ribonuclease action, and PCR amplification was performed using mouse repeat primers. The PCR products were cloned and used to screen a mouse genomic cosmid library, and the resultant cosmid clones were analyzed by fluorescence in situ hybridization. Ten of 70 clones analyzed gave telomere-proximal hybridization signals, indicating an at least 500-fold enrichment for telomere-proximal sequences.
Rounds
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Yale University School of Medicine, Department of Molecular Biophysics and Biochemistry, New Haven, Connecticut 06510, USA.
Brueckner
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Ward
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D000818:Animals; D001360:Avidin; D001483:Base Sequence; D016384:Consensus Sequence; D003360:Cosmids; D004247:DNA / Q000032:analysis; D017931:DNA Primers; D015342:DNA Probes; D015252:Deoxyribonucleases, Type II Site-Specific; D005090:Exodeoxyribonucleases; D015698:Genomic Library; D017404:In Situ Hybridization, Fluorescence; D007621:Karyotyping; D051379:Mice; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000032:analysis; D012091:Repetitive Sequences, Nucleic Acid; D016615:Telomere; D014764:Viral Proteins
D017931:DNA Primers; D015342:DNA Probes; D014764:Viral Proteins; D001360:Avidin; D012313:RNA; D004247:DNA; D005090:Exodeoxyribonucleases; C043586:exo protein, Bacteriophage lambda; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases