Semiautomated DNA probe mapping using digital imaging microscopy: II. System performance

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Abstract
This paper describes an evaluation of a semiautomated, multicolor image‐analysis system to map cloned probes along metaphase chromosomes. Mapping with this system consists of fluorescence in situ hybridization (FISH) for probe localization, automatic acquisition of multicolor images showing total chromosomal DNA and probe location(s), and automatic determination of the fractional locations of the probes along the chromosomes relative to the short arm telomere (FLpter). The system was evaluated by mapping ten phage and ten cosmid probes previously mapped to chromosome 3 with other procedures. The standard deviations of FLpter measurements averaged 3.4 Mb and 2.6 Mb for phage and cosmid probes, respectively. With this variation, the order of two probes mapped in separate hybridizations could be determined with 95% confidence when their separation was greater than 2.5 Mb. In all cases, the probe locations and order were consistent with previous mapping data. FLpter values were converted to band locations using measurements of the band locations made using digital imaging microscopy. This proved superior to conversions made using ISCN ideograms.