A method for the rapid generation of alpha- and classical satellite probes for human chromosome 9 by polymerase chain reaction using genomic DNA and their application to detect chromosomal alterations in interphase cells.

Abstract

Fluorescence in situ hybridization (FISH) using chromosome-specific DNA probes is a technique which has recently become widely used for the analysis of chromosome alterations in interphase and metaphase cells. In this report, a polymerase chain reaction (PCR)-based method is described for simultaneously amplifying and labelling probes targeting the alpha- and classical satellite regions of chromosome 9 using either plasmid or genomic DNA. Chromosome-specific probes were generated using readily obtainable plasmid DNA and genomic DNA from a hybrid cell line containing human chromosome 9 in a hamster cell background. The utility of these probes to detect and quantify structural and numerical aberrations in interphase cells was demonstrated using a new multicolor FISH strategy by comparing the frequencies of hyperdiploidy and chromosome breakage affecting the regions targeted by the probes in interphase and metaphase human lymphocytes irradiated during culture. The irradiated cells exhibited a significantly higher frequency of tetrasomy and breakage effecting the centromeric/pericentric region of chromosome 9 as compared with non-exposed cells. In general, similar frequencies of breakage and hyperdiploidy were observed in the interphase and metaphase preparations. These results show that DNA probes for the repetitive sequences in human chromosomes can be easily generated from genomic DNA and that these probes can be effectively used to detect chromosome breakage and aneuploidy in interphase and metaphase lymphocytes in vitro.