In situ detection of a PCR-synthesized human pancentromeric DNA hybridization probe by color pigment immunostaining: Application for dicentric assay automation

We report a low cost and efficient method for synthesizing a human pancentr omeric DNA probe by the polymerase chain reaction (PRC) and an optimized pr otocol for in situ detection using color pigment immunostaining, The DNA te mplate used in the PCR was a 2.4 kb insert containing human alphoid repeate d sequences of pancentromeric DNA subcloned into pUC9 (Miller ct al, 1988) and the primers hybridized to internal sequences of the 172 bp consensus ta ndem repeat associated with human centromeres. PCR was performed in the pre sence of biotin-11-dUTP, and the product was used for in situ hybridization to detect the pancentromeric region of human chromosomes in metaphase spre ads. Detection of pancentromeric probe was achieved by immunoenzymatic colo r pigment painting to yield a permanent image detected at high resolution b y bright field microscopy, The ability to synthesize the centromeric probe rapidly and to detect it with color pigment immunostaining will lead to enh anced identification and eventually to automation of various chromosome abe rration assays.