RAPID GENE DISCOVERY

描述：(申请人摘要)：项目2(快速基因发现)具有AS 它的主要目标是发现被……干扰的基因(S)。 个体平衡染色体异常与发育相关 异常现象。我们将收到来自项目1(高吞吐量鱼类)的BAC克隆 映射)跨越平衡像差的位置和来自 相应的病人。断点将按优先顺序进行分析，以 最大限度地增加突变与患者表型有关的可能性 或者，接近休止期的基因可以被有效地评估为候选基因。vbl.使用 来自人类基因组序列的基于PCR的策略，我们将与 项目1关于检测染色体断裂点的位置 BAC。改变的染色体序列将与 相应的正常序列以确定任何缺失的程度， 断点处的复制或其他重排。我们将确定基因 在每条染色体上的断裂点附近通过组合 数据库搜索，跨物种比较，计算基因预测， 逆转录-聚合酶链式反应、聚合酶链式反应和杂交法的文库筛选。基因 被断点破坏的将由cDNAFISH(项目1)和 用cDNA探测患者DNA的Southern斑点。我们将使用Northern印迹和 RT-PCR分析以评估被破坏的基因是否产生新的转录本 并将扩展选定的cDNA以定义完整的编码序列、筛选 这些是可能预测基因功能的蛋白质基序或同源序列。哪里 可能的、被破坏的基因或位置效应导致的失控候选基因 将分析一组独立的非易位突变 表现出相似表型的患者。我们的目标是初步分析8 每年的单个断点，从项目1的染色体中提取 直接或通过此项目的帮助确定“跨越”BAC 用于在GenBank或BAC文库中进行基因组行走。项目2中发现的基因 将被转发到项目3以分析发育表达和 根据最具吸引力的选择的所有可用数据确定优先顺序 模型系统中的功能遗传分析。

DESCRIPTION: (Applicant's Abstract): Project 2 (Rapid Gene Discovery) has as its major goal the discovery of the gene(s) disrupted by or in the vicinity of individual balanced chromosome aberrations associated with developmental abnormalities. We will receive BAC clones from Project 1 (High Throughput FISH Mapping) that cross the sites of balanced aberrations and DNA from the corresponding patients. Breakpoints will be prioritized for analysis to maximize the likelihood that the break is responsible for the patient phenotype or that genes near the break can be effectively assessed as candidates. Using PCR-based strategies from the human genomic sequence, we will collaborate with Project 1 on detection of the site of the chromosomal breakpoint within the BAC. The sequence of altered chromosomes will be compared with the corresponding normal sequences to determine the extent of any deletion, duplication or other rearrangement at the breakpoints. We will identify genes in the vicinity of the breakpoint on each chromosome by a combination of database searching, cross-species comparison, computation gene prediction, RT-PCR and PCR-based and hybridization-based cDNA library screening. Genes disrupted by the breakpoint will be identified by cDNA FISH (Project 1) and by probing Southern blots of patient DNA with cDNAs. We will use Northern blot and RT-PCR analysis to assess whether the disrupted genes produce novel transcripts and will extend selected cDNAs to define the full coding sequence, screening these for protein motifs or homologies that might predict gene function. Where possible, disrupted genes or candidates for disregulation by position effects will be analyzed for mutations in an independent set of non-translocation patients displaying similar phenotypes. Our goal is to initially analyze 8 individual breakpoints per year, drawn from the chromosomes for which Project 1 identifies "crossing" BACs either directly, or through the help of this project for genomic walking in GenBank or a BAC library. Genes discovered in Project 2 will be forwarded to Project 3 for analysis of developmental expression and prioritization based on all available data of the most compelling choices for functional genetic analysis in model systems.