COMPARATIVE HYBRIDIZATION OF AP PCR ARRAYS

AP PCR 阵列的比较杂交

• 批准号: 6522342
• 负责人: Sergei R Malkhosyan
• 金额: $ 40.62万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-15 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6522342
• 关键词: DNA; biopsy; fluorescent dye /probe; genome; human tissue; microarray technology; neoplasm /cancer genetics; nucleic acid hybridization; oligonucleotides; polymerase chain reaction; restriction fragment length polymorphism

DESCRIPTION: (Applicant's Description) We applied an unbiased DNA fingerprinting technique, the Arbitrarily-Primed PCR (AP-PCR) to study tumor- specific genetic changes. AP-PCR is a PCR-based DNA fingerprinting method, which uses a single oligonucleotide primer of arbitrary sequence, and generates a profile of quantitative and qualitative differences between the fingerprints of tumor and matched-normal tissues. Our efforts to both automate the AP-PCR technique and make it more robust, gave us the idea of combining AP-PCR fingerprinting with DNA array hybridization technology. Developing this new technique, which we call Comparative Hybridization of AP- PCR Arrays (CHAPA), is the goal of this grant application. We propose to clone individual DNA fragments amplified by AP-PCR from human genomic DNA and array them on a solid base. The AP-PCR product can be viewed as a low- complexity representation of the genome. The array will be hybridized with AP-PCR products that are amplified from normal and tumor tissue DNAs, which have been labeled with green and red fluorescent dyes, respectively. The intensity ratio of the two colors at each hybridization spot will reflect tumor-specific losses, gains, or no change of corresponding genomic loci. For the Phase I application, we will develop a small array of 300 AP-PCR fragments and test the new technique to see if it is reproducible, sensitive, and reliable. Chromosomal and subchromosomal origins of the arrayed AP-PCR fragments will be determined by hybridizing the array with AP-PCR products from individual clones of radiation hybrid mapping panels. We will also test different arbitrary primers to obtain collections of AP-PCR products (representations) of the human genome with a degree of complexity that is optimal for a large scale array of AP-PCR fragments. We will also test AP- PCR's ability to produce quantitative fingerprints of genomic DNA isolated from minute amounts of fixed microdissected tissues. For the Phase II experiments, we propose to scale up the array to at least 5,000 nonredundant AP-PCR fragments. In its final form, the technique will allow the state of the tumor genome to be quickly and automatically analyzed with less than 1 Mbp of resolution. This high-density unbiased molecular karyotyping will facilitate the discovery of novel cancer genes and open new horizons for the diagnostic and prognostic analysis of cancer development.

描述：（申请人的描述）我们应用了公正的 DNA 指纹技术、任意引物 PCR (AP-PCR) 来研究肿瘤 特定的基因变化。 AP-PCR是一种基于PCR的DNA指纹分析方法， 它使用任意序列的单个寡核苷酸引物，并且 生成定量和定性差异的概况 肿瘤和匹配的正常组织的指纹。 我们的努力 自动化 AP-PCR 技术并使其更加稳健，给了我们这样的想法 将 AP-PCR 指纹识别与 DNA 阵列杂交技术相结合。 开发这种新技术，我们称之为 AP 的比较杂交 PCR 阵列 (CHAPA) 是本次拨款申请的目标。 我们建议 从人类基因组 DNA 中克隆通过 AP-PCR 扩增的单个 DNA 片段， 将它们排列在坚固的底座上。 AP-PCR 产物可被视为低 基因组的复杂性表示。 该阵列将与 从正常和肿瘤组织 DNA 中扩增出的 AP-PCR 产物， 分别用绿色和红色荧光染料标记。 这 每个杂交点处两种颜色的强度比将反映 肿瘤特异性相应基因组位点的丢失、增加或无变化。 为了 在第一阶段的应用中，我们将开发一个包含 300 个 AP-PCR 片段的小阵列 并测试新技术，看看它是否可重复、是否灵敏以及 可靠的。 阵列 AP-PCR 的染色体和亚染色体起源 通过将阵列与 AP-PCR 产物杂交来确定片段 来自辐射混合绘图面板的单个克隆。 我们也会测试 不同的任意引物以获得AP-PCR产物的集合 （表示）人类基因组的复杂程度为 最适合大规模 AP-PCR 片段阵列。 我们还将测试 AP- PCR 能够产生分离的基因组 DNA 的定量指纹 来自微量固定的显微解剖组织。 对于第二阶段 实验中，我们建议将阵列扩展到至少 5,000 个非冗余 AP-PCR 片段。 在其最终形式中，该技术将允许以下状态 以小于 1 Mbp 的速度快速自动分析肿瘤基因组 的分辨率。 这种高密度无偏见的分子核型分析将 促进新型癌症基因的发现，为癌症研究开辟新视野 癌症发展的诊断和预后分析。