COMPARATIVE HYBRIDIZATION OF AP PCR ARRAYS

AP PCR 阵列的比较杂交

• 批准号: 6496631
• 负责人: Sergei R Malkhosyan
• 金额: $ 39.72万
• 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 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6496631
• 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，任意培养的PCR（AP-PCR）研究肿瘤 特定的遗传变化。 AP-PCR是一种基于PCR的DNA指纹方法， 它使用任意序列的单个寡核苷酸引物，并且 产生定量和定性差异的概况 肿瘤和匹配正常组织的指纹。 我们为俩所做的努力 自动化AP-PCR技术并使其更强大，使我们的想法 将AP-PCR指纹与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）是此赠款申请的目标。 我们建议 克隆个体DNA片段通过人类基因组DNA和 在坚实的基础上阵列。 AP-PCR产品可以看作是低 - 基因组的复杂性表示。 阵列将与 从正常和肿瘤组织DNA放大的AP-PCR产品， 分别用绿色和红色荧光染料标记。 这 每个杂交点的两种颜色的强度比将反映 肿瘤特异性损失，增长或不变相应的基因组基因座。 为了 I阶段应用程序，我们将开发一个300个AP-PCR片段的小数组 并测试新技术以查看它是否可重现，敏感和 可靠的。 ARDED AP-PCR的染色体和亚染色体起源 片段将通过将阵列与AP-PCR产品杂交确定 来自辐射混合映射面板的单个克隆。 我们还将测试 不同的任意引物以获取AP-PCR产品的收集 人类基因组的（表示），具有一定程度的复杂性 最佳的AP-PCR片段阵列。 我们还将测试AP- PCR产生分离基因组DNA的定量指纹的能力 从微量的固定微分辨率组织。 对于II期 实验，我们建议将阵列扩展到至少5,000个非冗余 AP-PCR片段。 以最终形式，该技术将允许 肿瘤基因组将快速并自动用小于1 MBP进行分析 解决方案。 这种高密度的无偏分子核分型将 促进发现新颖的癌症基因并开放新的视野 癌症发展的诊断和预后分析。