The overall goal of this application is to develop analytical cytology and fluorescence in situ hybridization for improved prenatal detection of genetic disease.
Specific aims i nclude: 1) Final optimization of bivariate flow karyotyping for detection of numerical and structural chromosome aberrations. After optimization, flow karyotyping will be applied to study the heritability of normal chromosome polymorphisms and to detection of clinically important structural chromosome aberrations that involve changes in chromosomal DNA content or base composition of less than 0.5%. 2) Development of fluorescence in situ hybridization (called fluorescence hybridization) and chromosome- specific-nucleic acid probes to allow identification and enumeration of human chromosomes 21, 18, 13, X and Y in metaphase and interphase cells taken for prenatal diagnosis (ie. during amniocentesis or chorionic villus biopsy). Chromosome specific nucleic acid probes will be either; collections of unique sequence probes selected from recombinant DNA libraries made using DNA from a single chromosome, or chromosome specific repeat sequence probes. The unique sequence probe collections will be designed to produce uniform fluorescence hybridization along the length of a chromosome. The repeat sequence probes will allow fluorscence hybridization to limited chromosome regions (eg. to chromosome centromeres). These studies will facilitate detection of aberrant chromosomes by allowing more distinctive staining of selected metaphase chromosomes and/or by allowing detection of aneuploidy in interphase cells thereby eliminating time consuming cell culture. 3) Development of analytical cytologic procedures for selection of fetal cells from maternal blood and for detection of chromosomal aneuploidy involving chromosomes 21, 18, 13, X and Y in interphase fetal cells from maternal blood using fluorescence hybridization. These studies should firmly establish the presence of fetal cells in maternal blood, allow determination of their frequency as a function of fetal age and lead to a non-invasive procedure for fetal cytogenetic analysis.
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