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）我们应用了无偏的DNA 指纹技术，引物PCR（AP-PCR）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， 其使用任意序列的单一寡核苷酸引物，和 生成一个配置文件之间的定量和定性差异， 肿瘤和匹配的正常组织的指纹。 我们的努力， 使AP-PCR技术自动化并使其更强大，给了我们这样的想法， 结合AP-PCR指纹图谱和DNA阵列杂交技术。 开发这种新技术，我们称之为AP的比较杂交， PCR阵列（CHAPA），是这个赠款申请的目标。 我们建议 从人基因组DNA中克隆通过AP-PCR扩增的单个DNA片段， 把它们排列在坚实的基础上。 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扩增的AP-PCR产物， 分别用绿色和红色荧光染料标记。 的 在每个杂交点处的两种颜色的强度比将反映 肿瘤特异性损失、获得或相应基因组基因座无变化。 为 在第一阶段的应用中，我们将开发一个300个AP-PCR片段的小阵列， 并测试新技术，看看它是否是可重复的，敏感的， 可靠 阵列式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 从辐射杂交绘图板的单个克隆中。 我们还将测试 不同的随机引物，以获得AP-PCR产物的集合 人类基因组的复杂程度， 最适合于大规模的AP-PCR片段阵列。 我们还将测试AP- PCR产生分离的基因组DNA的定量指纹的能力 从微量的固定的显微组织中分离出来。 II期 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 肿瘤基因组快速自动分析，小于1 Mbp 的决议。 这种高密度无偏倚的分子核型分析将 促进新的癌症基因的发现，并为癌症研究开辟新的视野。 癌症发展的诊断和预后分析。