Chromosome-specific DNA repeat probes.
In research as well as in clinical applications, fluorescence in situ hybridization (FISH) has gained increasing popularity as a highly sensitive technique to study cytogenetic changes. Today, hundreds of commercially available DNA probes serve the basic needs of the biomedical research community. Widespread applications, however, are often limited by the lack of appropriately labeled, specific nucleic acid probes. We describe two approaches for an expeditious preparation of chromosome-specific DNAs and the subsequent probe labeling with reporter molecules of choice. The described techniques allow the preparation of highly specific DNA repeat probes suitable for enumeration of chromosomes in interphase cell nuclei or tissue sections. In addition, there is no need for chromosome enrichment by flow cytometry and sorting or molecular cloning. Our PCR-based method uses either bacterial artificial chromosomes or human genomic DNA as templates with alpha-satellite-specific primers. Here we demonstrate the production of fluorochrome-labeled DNA repeat probes specific for human chromosomes 17 and 18 in just a few days without the need for highly specialized equipment and without the limitation to only a few fluorochrome labels.
Baumgartner
Adolf
A
Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, California, CA 94720, USA.
Weier
Jingly Fung
JF
Weier
Heinz-Ulrich G
HU
D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D002886:Chromosomes, Human, Pair 17 / Q000235:genetics*; D002887:Chromosomes, Human, Pair 18 / Q000235:genetics*; D003001:Cloning, Molecular; D015342:DNA Probes; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012091:Repetitive Sequences, Nucleic Acid; D012680:Sensitivity and Specificity
D015342:DNA Probes