NEW METHODS FOR ANALYZING HUMAN GENES AND THEIR DEFECTS

本提案的总体目标是：1）继续发展 分析人类染色体的新方法和DNA探针， 正常和肿瘤细胞或组织中的基因，2）实施 多参数荧光成像技术，可应用于 同时分析多个染色体、基因、mRNA或蛋白质， 多样的生物标本，以及3）遗传变化的检查 发生于头颈部鳞状细胞癌。 具体 研究目标是：1.开发一种自动化的染色体分析方法， 基于荧光原位杂交（FISH）的核型分析。 24个染色体特异性“绘画”探针库和一个RT-PCR “显带”探针将同时与染色体杂交 注意，每个探针组将标记五种探针中的一种或多种 光谱上不同的荧光体，当用于简单的布尔组合时， 将区分所有25个探针。 数字成像显微镜将有 所有阶段的扫描操作，滤光片变化，光源荧光 图像采集、CCD数据下载和探头识别， 在计算机控制下的光谱特征 将在盲态中使用细胞遗传学定义的染色体异常。 时尚，测试、改进和比较FISH核型分析与传统的 细胞遗传学 一旦确认，将使用FISH进行核型分析 从卵巢和乳腺的短期培养物制备染色体 通过针吸和活组织检查获得的肿瘤组织。 2.评价 优化染色体、基因、mRNA和 蛋白质在培养的细胞和新鲜冷冻或甲醛固定， 石蜡包埋组织，通过确定可以分析多少探针 通过定量色谱法明确分离 荧光显微镜，并建立在何种程度上，这是 受探针大小、探针的遗传复杂性或拷贝数的影响 目标分析物。 3.以确定重大的遗传异常， 基因剂量测定法治疗头颈部鳞状细胞癌 异常（非整倍体、杂合性丢失或基因扩增） 通过比较个体中发现的基因型异常， 多发性原发癌与存在于单个 原发性病变 遗传分析将通过以下组合进行： 比较基因组杂交（CGH），FISH，杂合性丢失（洛） 使用PCR扩增的多态性微卫星标记进行的研究， 分析核苷酸重复序列的潜在扩展。 肿瘤选择 来自耶鲁大学组织库的标本和基因型数据的评估， 临床与肿瘤分期、患者生存率或 转移性疾病将与机构合作进行 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.