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

被引:6
作者
ROUNDS, D
BRUECKNER, M
WARD, DC
机构
[1] YALE UNIV,SCH MED,DEPT PEDIAT CARDIOL,NEW HAVEN,CT 06510
[2] YALE UNIV,SCH MED,DEPT BIOCHEM & MOLEC BIOPHYS,NEW HAVEN,CT 06510
[3] YALE UNIV,SCH MED,DEPT GENET,NEW HAVEN,CT 06510
关键词
D O I
10.1006/geno.1995.9958
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
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. (C) 1995 Academic Press, Inc.
引用
收藏
页码:616 / 622
页数:7
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