NEW METHODS FOR ANALYZING HUMAN GENES AND THEIR DEFECTS

本方案的总体目标是：1)继续发展 人类染色体和DNA分析的新方法和DNA探针 正常和肿瘤细胞或组织中的基因，2)实现 多参数荧光成像技术可应用于 多条染色体、基因、mRNAs或蛋白质的同时分析 多样化的生物标本，以及3)遗传变化的检查 发生于头颈部的鳞状细胞癌。特定的 研究目标是：1.开发一种自动化的染色体检测方法 基于多色荧光原位杂交的核型分析。 24个染色体特异的“涂色”探针库和Alu-PCR “显带”探针将同时杂交到染色体上 准备，每个探针组将被标记为五个中的一个或多个 当在简单的布尔组合中使用时， 将区别对待所有25个探测器。数字成像显微镜将具有 所有阶段扫描操作、滤光片更换、源荧光 图像采集、CCD数据下载和探头识别 计算机控制下的光谱签名细胞系包含 细胞遗传学定义的染色体异常将在盲法中使用 时尚，测试，改进和比较鱼类核型和常规 细胞遗传学。一旦验证，多色FISH将用于核型分析 卵巢和乳腺短期培养中的染色体制备 通过针吸和活检获得的肿瘤组织。2.评估 并优化了染色体、基因、信使核糖核酸和 培养细胞和新鲜冷冻或甲醛固定的蛋白质， 通过确定可以分析多少探针来检测石蜡包埋组织 定量多色同时、非等位分辨 荧光显微镜，并确定这在多大程度上 受探头大小、探头或拷贝数的磁复杂性的影响 目标分析物的数量。3.找出显著的遗传异常 确定基因剂量对头颈部鳞状细胞癌的影响 异常(非整倍体、杂合性丢失或基因扩增) 通过比较在个体中发现的基因异常和 多原发癌与单发单纯性癌 原发病变。基因分析将通过结合以下几种方法进行 比较基因组杂交、FISH、杂合性缺失 利用聚合酶链式反应扩增的多态微卫星标记的研究，以及 核苷酸重复序列的潜在扩展分析。肿瘤切除 来自耶鲁组织库的样本和对基因数据的评估 临床与肿瘤分期、患者生存或发生 转移性疾病将与机构合作完成 肿瘤学家和病理学家。

Th overall objectives of this proposal are 1) to continue the development of new methods and DNA probes for the analysis of human chromosomes and genes in normal and neoplastic cells or tissues, 2) the implementation of multiparameter fluorescence imaging technology that can be applied to the simultaneous analysis of multiple chromosomes, genes, mRNAs or proteins in diverse biological specimens, and 3) the examination of genetic changes occurring in squamous cell carcinomas of the head and neck. Specific research goals are: 1. to develop an automated method of chromosome karyotyping based on multicolor fluorescence in situ hybridization (FISH). Twenty-four chromosome-specific "painting" probe libaries and an Alu-PCR "banding" probe will be hybridized simultaneously to chromosome preparatuibs, Each probe set will be labeled with one or more of five spectrally distinct fluors which, when used in simple boolean combination, will discriminate all 25 probes. The digital imaging microscope will have all stage scanning operations, optical filter changes, source fluorescence image capture, CCD data downloading and identification of probes by spectral signature under computer control Cell lines containing cytogenetically defined chromosomal abnormalities will be used, in a blind fashion, to test, refine and compare FISH karyotyping with conventional cytogenetics. Once validated, multicolor FISH will be used to karyotype chromosome preparations from short-term cultures of ovarian and breast tumor tissues obtained by needle aspiration and biopsy. 2. to evaluate and optimize the multiplex analysis of chromosomes, genes, mRNA and proteins in cultured cells and fresh frozen or formaldehyde-fixed, paraffin-embedded tissues by determining how many probes can be analyzed simultanelusly and resolved unequiocally by quantitative multicolor fluorescence microscopy, and to establish the extent to which this is influenced by probe size, gnetic complexity of the probe or the copy nmber of the target analytes. 3. to identify significant genetic anomalies in squamous cell carcinomas of the head and neck by determining gene dosage abnormalities (aneuploidy, loss of heterozygosity or gene amplification) and by comparing the genotypic anomalies found in individuals with multiple primary carcinomas with those present in individuals with single primary lesions. Genetic analysis will be done by a combination of comparative genome hybridization (CGH), FISH, loss of heterozygosity (LOH) studies using polymorphic microsatellite markers amplified by PCR, and analysis of potential expensions of nucleotide repeats. Slection of tumor specimens from the YALE tissue bank and evaluation of genotypic data for clinical correlates with tumor staging, patient survival or occurrence of metastatic disease will be done in collaboration wit institutional oncologists and pathologists.