FLOW KARYOTYPING: OPTIMIZATION AND CLINICAL EVALUATION

该项目的总体目标是开发和评估改进 分析细胞学技术用于疾病相关疾病的产前检测 细胞遗传学异常。重点将放在a)提高速度和 可以检测到染色体DNA丢失或获得的灵敏度 并在中期染色体分析期间进行量化，b)检测 间期细胞常见的细胞遗传学异常与c)发育 应用分离的胎儿细胞进行细胞遗传学分析的技术 母血。这些目标将通过以下方式实现： 1)应用双变量Flow染色体核型技术定量研究 小到1Mb的染色体缺失和复制。在此过程中的努力 项目期将致力于进一步提高 技术，并将其应用于一系列合作研究中 描述临床或分子的细微结构异常 生物学上的重要性。 2)优化染色体特异的荧光原位杂交 用于可靠检测和表征数值像差的探头 涉及21、18、13、x和y染色体；节段性复制 (最初为唐氏综合症21q22.3)和删除(最初为15q11-13 Prader-Willi综合征)，在中期扩散和间期核。 3)开发染色体特异的探针以支持荧光 杂交研究。全染色体DNA和单链RNA探针耗尽 重复序列将从染色体特定的粘粒中产生 图书馆。21q22.3、15q11-13和其他地方的复合探测器将 通过从已映射到这些区域的“种子”探测开始生成 并在染色体特异的粘粒文库中向外“行走”，以扩展 复合探头覆盖的区域。 4)继续努力从母亲血液中分离胎儿细胞 间期细胞遗传学分析。具体项目包括：a) 确定妊娠头三个月内的孕周 胎儿细胞在母体中出现的频率最高 血液，b)确定胎儿细胞的来源(例如，白细胞或 细胞滋养层细胞)和c)程序的开发 胎儿细胞。

The overall objective of this project is to develop and evaluate improved analytical cytologic techniques for prenatal detection of disease-linked cytogenetic abnormalities. Emphasis will be on a) improving the speed and sensitivity with which the loss or gain of chromosomal DNA can be detected and quantified during analysis of metaphase chromosomes, b) detection of common cytogenetic abnormalities in interphase cells and c) developing techniques for cytogenetic analysis using fetal cells isolated from maternal blood. These objectives will be pursued by: 1) Applying bivariate flow karyotyping to quantification of the extent of chromosome deletions and duplications as small as 1 Mb. Effort during this project period will be devoted to further increasing the sensitivity of the technique and applying it in a series of collaborative studies to characterize subtle structural aberrations of clinical or molecular biological importance. 2) Optimizing fluorescence in situ hybridization with chromosome specific probes for reliable detection and characterization of numerical aberrations involving chromosomes 21, 18, 13, x and y; segmental duplications (initially 21q22.3 for Down syndrome) and deletions (initially 15q11-13 for Prader-Willi syndrome) in metaphase spreads and in interphase nuclei. 3) Developing chromosome-specific probes to support the fluorescence hybridization studies. Whole-chromosome DNA and ssRNA probes depleted in repetitive sequences will be generated from chromosome-specific cosmid libraries. Composite probes for 21q22.3, 15q11-13 and elsewhere will be generated by starting with "seed" probes already mapped to these regions and "walking" outward in chromosome-specific cosmid libraries to expand the regions covered by the composite probes. 4) Continuing efforts to isolate fetal cells from maternal blood for interphase cytogenetic analysis. Specific projects include: a) determination of the gestational age within the first trimester at which the fetal cells are present at highest frequency in maternal blood,b)determination of the origin of the fetal cells (e.g. leukocytes or cytotrophoblasts) and c) development of procedures for enrichment of the fetal cells.