EFFICIENT GENE MAPPING BY WHOLE GENOME MISMATCH SCANNING

通过全基因组错配扫描进行高效基因图谱

• 批准号: 3443850
• 负责人: PATRICK O. BROWN
• 金额: $ 12.83万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3443850
• 关键词: Drosophilidae; alleles; autosomal recessive trait; biochemical evolution; endonuclease; extrachromosomal DNA; fluorescent dye /probe; genetic mapping; genome; human genetic material tag; human population genetics; human tissue; in situ hybridization; linkage mapping; molecular genetics; nucleic acid sequence

The goal of the proposed work is to develop a radically new method for rapid direct mapping of the regions of genetic identity-by-descent between two related individuals, using only DNA samples from the two individuals. The method is intended to allow the simple and powerful but presently impractical "affected relative pair" mapping strategy to be put into practice. The principal advantages of the proposed method are: 1. No other family members need to be examined, and no pedigree information is required. 2. No locus-specific probes are required. 3. The entire genetic map is surveyed at high resolution in a single procedure, rather than through multiple discrete analyses of polymorphic loci at intervals throughout the genetic map. The method takes advantage of the fact that (at least in an outbred Western European or US population) any two allelic sequences that are not inherited from a common recent ancestor have single base differences on average every few hundred (about 300) basepairs. In other words, for an arbitrary pair of relatives, there are around 10-7 potentially informative "sequence polymorphisms" per haploid genome. The proposed method is designed to provide a practical way to exploit this vast source of information for gene mapping. The method involves four steps: 1. Preparing genomic DNA from two related individuals. 2. Preparing and isolating hybrid DNA molecules containing one strand from each individual. 3. Selecting those DNA hybrids that are free of mismatches over many thousands of base-pairs, and therefore very likely to represent sites of genetic identity by descent. 4. Using the resulting large, heterogeneous pool of perfectly, matched DNA hybrids as the template for probes, initially for in situ hybridization to metaphase chromosomes, to identify the map locations of sequences identical by descent between the two tested individuals. With a sufficiently large collection of affected relative pairs, the frequency of genetic concordance at the map position(s) of the trait-determining gene(s) will exceed that expected to occur by chance. In the case of a rare trait uniquely determined by a dominant allele of a single gene (e.g., von Recklinhausen's neurofibromatosis), 10 pairs of relatives sharing the trait would generally be sufficient to map the gene to a single 10 megabase region of the genome. Most of the technical obstacles to implementation of this approach have actually been overcome by previous workers, in other contexts. Thus, while the proposed concatenation and adaptation of available techniques to gene mapping represents a fundamental departure from previous mapping strategies, no fundamental technical breakthroughs are invoked. A closely-related method for mapping rare recessive traits using DNA from only one or a few affected individuals will also be tested.

拟议工作的目标是开发一种全新的方法 快速直接绘制不同血统之间的遗传同一性区域 两个相关的个体，仅使用两个个体的 DNA 样本。 该方法旨在允许简单而强大但目前 不切实际的“受影响亲属对”映射策略 实践。 所提出方法的主要优点是： 1. 不需要 其他家庭成员需要检查，并且没有血统信息 必需的。 2. 不需要位点特异性探针。 3.全基因 通过单一程序以高分辨率测量地图，而不是 通过对多态位点进行多次离散分析 贯穿整个遗传图谱。 该方法利用了以下事实： （至少在近亲繁殖的西欧或美国人群中）任何两个等位基因 不是从共同的最近祖先继承的序列具有单一的 平均每几百（约 300）个碱基对就有碱基差异。 在 换句话说，对于任意一对亲戚，大约有 10-7 个 每个单倍体基因组的潜在信息“序列多态性”。 这 所提出的方法旨在提供一种实用的方法来利用这一巨大的 基因图谱的信息来源。 该方法包括四个步骤： 1. 制备两个相关个体的基因组 DNA。 2. 准备和 从每个个体中分离出含有一条链的杂交 DNA 分子。 3. 选择那些没有错配的 DNA 杂交体 数千个碱基对，因此很可能代表 血统的遗传同一性。 4. 使用由此产生的大型、异构的数据 完美匹配的 DNA 杂交体库作为探针的模板， 最初用于中期染色体的原位杂交，以鉴定 两个测试之间血统相同的序列的图谱位置 个人。 拥有足够多的受影响亲属 对，在图谱位置上遗传一致性的频率 性状决定基因将超出预期的偶然发生。 在 由显性等位基因唯一决定的稀有性状的情况 单个基因（例如，von Recklinhausen 神经纤维瘤病），10 对 具有相同特征的亲属通常足以绘制基因图谱 到基因组的单个 10 兆碱基区域。 大部分是技术性的 实际上已经克服了实施这一方法的障碍 以前的工人，在其他情况下。 因此，虽然提议的 现有技术的串联和适应基因图谱 代表了与以前的绘图策略的根本背离，没有 引发根本性技术突破。 密切相关的方法 用于使用仅来自一个或几个受影响者的 DNA 绘制罕见的隐性特征 个人也将接受测试。