IDENTIFICATION OF A DNA MARKER LINKED TO CYSTIC FIBROSIS

与囊性纤维化相关的 DNA 标记的鉴定

• 批准号: 3233188
• 负责人: LAP-CHEE TSUI
• 金额: $ 7.43万
• 依托单位: HOSPITAL FOR SICK CHLDRN (TORONTO)
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-01-01 至 1987-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3233188
• 关键词: autoradiography; autosomal recessive trait; biological polymorphism; cystic fibrosis; endonuclease; gel electrophoresis; genetic disorder diagnosis; genetic manipulation; genetic markers; human population genetics; human subject; information systems; linkage mapping; molecular genetics; molecular pathology; nucleic acid sequence; prenatal diagnosis; tissue /cell culture

The long term objective of our research program is to define the basic defect in Cystic Fibrosis(CF). The approach described in this application will initially bypass the search for a specifid defect in CF. Instead, our specific aim is to identify a DNA marker closely linked to the disease locus. The study exploits the DNA sequence heterogeneity prevalent in the human population. Since these hereditary sequence polymorphisms are conveniently detected by using restriction endonucleases, the variable lengths of specifc DNA fragments generated from enzyme digestions have been used as markers in genetic analysis. By following the inheritance of a polymorphic DNA marker in families with CF patients, statistical methods are available to determine whether the disease is linked to the marker. A systematic screening of DNA markers distributed over the entire genome (excluding the sex chromosomes) should lead to the discovery of one such marker closely linked to the CF gene. This approach is expected to yield positive results because it is designed to examine the patient's genome directly rather than measuring parameters which could be due to secondary manifestation of the basic defect. Based on the size of the human genome and recombination frequencies, it has been estimated that approximately 150 to 400 markers mikght be required to map an unknown locus. However, this just sets the upper limit of the number of probes to be analyzed. In fact, a marker closely linked to Huntington Disease was found after screening only 12 random markers. To pursue our study, two-generation families with two or more CF children have been chosen as panels for screening DNA markers. DNA samples are prepared from lymphoblast cell lines established from each family member, digested with appropriate restriction enzymes, size-fractionated by agarose gel electrophoresis, transferred to DNA-binding membranes and hybridized with radioactively labelled DNA probes. The restriction fragment length polymorphisms are then revelaed by autoradiography. To facilitate analysis and storage of data we have developed computer-assisted systems. The discovery of a CF-linked DNA marker will likely provide an opportunity for carrier detection and prenatal diagnosis. More importantly, however, the marker should eventually permit identification of the CF gene. Identification of the basic biochemical defect in CF is a prerequisite for the development of effective therapy for this disorder.

我们研究计划的长期目标是确定基本的 囊性纤维化（CF）缺陷。 本申请中描述的方法 最初将绕过对 CF 中特定缺陷的搜索。 相反，我们的 具体目标是确定与疾病密切相关的 DNA 标记 轨迹。 该研究利用了人类中普遍存在的 DNA 序列异质性 人口。 由于这些遗传序列多态性很容易 通过使用限制性核酸内切酶检测，可变长度 酶消化产生的特定 DNA 片段已被用作 遗传分析中的标记。 通过遵循多态性的继承 CF患者家族DNA标记，有统计方法 以确定疾病是否与标记物相关。 对分布在整个基因组中的 DNA 标记进行系统筛选 （不包括性染色体）应该导致发现这样一个 与CF基因紧密连锁的标记。 这种方法预计会产生 积极的结果，因为它旨在检查患者的基因组 直接而不是测量参数，这可能是由于二次 基本缺陷的表现。 基于人类基因组的大小 和重组频率，据估计大约 150 绘制未知基因座可能需要 400 个标记。 然而，这 只是设置要分析的探针数量的上限。 实际上， 筛选后发现与亨廷顿病密切相关的标记 只有 12 个随机标记。 为了继续我们的研究，有两个或更多 CF 孩子的两代家庭 已被选为筛选 DNA 标记的面板。 DNA 样本是 由每个家庭成员建立的淋巴母细胞系制备， 用适当的限制性内切酶消化，用琼脂糖进行大小分级 凝胶电泳，转移至 DNA 结合膜并杂交 用放射性标记的 DNA 探针。 限制性片段长度 然后通过放射自显影术揭示多态性。 为了便于分析 和数据存储我们开发了计算机辅助系统。 CF 连接 DNA 标记的发现可能会提供一个机会 用于携带者检测和产前诊断。 然而更重要的是， 该标记最终应该能够识别 CF 基因。 识别 CF 的基本生化缺陷是治疗的先决条件 开发针对这种疾病的有效疗法。