Generation of highly specific DNA hybridization probes for chromosome enumeration in human interphase cell nuclei Isolation and enzymatic synthesis of alpha satellite DNA probes for chromosome 10 by primer directed DNA amplification

Weier, H.U.; Rosette, C.D.; Matsuta, M.; Zitzelsberger, H.F.; Matsuta, M.; Gray, J.W.

Methods in Molecular and Cellular Biology 4(6): 231-248, 1993-


Accession: 031554388

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This report describes the rapid generation of DNA probes for enumeration of specific chromosomes in human interphase cell nuclei. Specifically, we applied in vitro DNA amplification using the polymerase chain reaction with selected DNA templates for enrichment of repeated, chromosome 10-specific DNA. The resulting fragments contained members of human alpha satellite DNA family and were cloned into plasmid vectors for further selection of sequences with high specificity and copy number. These probes have a number of practical advantages over most other enumerator probes. We discuss probe production and labeling strategies and demonstrate the application of non-isotopically labeled probes for in situ hybridization to metaphase chromosomes, interphase cell nuclei, or tissue sections. Our hybridization results demonstrate for the first time that a single, cloned alphoid repeat monomer can successfully be used to delineate the centromeric heterochromatin on human chromosome 10. Finally, we present results from DNA sequence analysis comparing two chromosome 10 probe DNAs with a published sequence of an alphoid DNA probe for chromosome 12.