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 pancentromeric DNA probe by the polymerase chain reaction (PRC) and an optimized protocol for in situ detection using color pigment immunostaining. The DNA template used in the PCR was a 2.4 kb insert containing human alphoid repeated sequences of pancentromeric DNA subcloned into pUC9 (Miller et al. 1988) and the primers hybridized to internal sequences of the 172 bp consensus tandem repeat associated with human centromeres. PCR was performed in the presence of biotin-11-dUTP, and the product was used for in situ hybridization to detect the pancentromeric region of human chromosomes in metaphase spreads. Detection of pancentromeric probe was achieved by immunoenzymatic color pigment painting to yield a permanent image detected at high resolution by bright field microscopy. The ability to synthesize the centromeric probe rapidly and to detect it with color pigment immunostaining will lead to enhanced identification and eventually to automation of various chromosome aberration assays.

Fast-in situ hybridization and immunoenzymatic color pigment detection of mouse bone marrow micronucleus.

The development of a whole mouse genomic DNA probe coupled to color pigment painting detection methodology can accurately verify mouse micronuclei induced by chemicals or drugs leading to a lower probability of potential artifacts. Using color pigment painting detection of probes in conjunction with Wright's Giemsa counterstain instead of the current fluorescence detection technology ensures low cost, high resolution permanent documentation of slides for a particular test compound. The permanent color pigment-detected micronuclei and adjoining counterstain allows slides to be stored for future analysis without enhancing the signal or adding antifading agents that are associated with fluorescence detection. Combining innovative technology such as fast-in situ hybridization of DNA probes with immunoenzymatic color pigment detection provides rapid verification of true micronuclei (DNA containing) within 2-3 hr.

Application of the premature chromosome condensation assay in simulated partial-body radiation exposures: evaluation of the use of an automated metaphase-finder.

The premature chromosome condensation (PCC) assay has been proposed as a useful and rapid end point for biological dosimetry following accidental high-dose radiation overexposures. A major benefit of the PCC assay is that it does not require cells to divide for evaluation of cytogenetic damage. The PCC assay was performed on isolated human peripheral lymphocytes exposed in vitro to doses from 1 to 9 Gy of 250 kVp x-rays. The dose-response relationships of the frequency distribution and the yield of PCC fragments in cells were determined after one day of repair at 37 degrees C. A Qpcc approach, which involves the analysis of the yield of excess PCC fragments in damaged cells, was used to establish a dose-response calibration curve. This method is identical in concept to the Qdr technique introduced by Sasaki for partial-body exposure dose-estimates using asymmetrical chromosome aberrations (i.e., dicentrics and rings) in metaphase spreads of human lymphocytes. A simulated in vitro test of a partial-body exposure to a 6-Gy dose was performed. The results from this test provided dose estimates of 5.3 +/- 0.6, 4.7 +/- 0.6, 5.0 +/- 0.6 and 4.7 +/- 0.8 Gy for the 20, 30, 50 and 75 percent component of 6-Gy irradiated cells, respectively. An automated metaphase-finding system was evaluated for use with the PCC assay. This system helped to locate PCC spreads among the mitotic inducer Chinese hamster ovary (CHO) metaphase spreads, thereby facilitating rapid scoring of samples.(ABSTRACT TRUNCATED AT 250 WORDS)