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: 3443849
• 负责人: PATRICK O. BROWN
• 金额: $ 8.64万
• 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/3443849
• 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.

这项工作的目标是开发一种全新的方法， 快速直接绘制遗传特征区域， 两个相关的个体，仅使用来自两个个体的DNA样本。 该方法旨在允许简单和强大的，但目前 不切实际的“受影响的相对对”映射策略， 实践 所提出的方法的主要优点是：1。没有 其他家庭成员需要检查，没有谱系信息， 必需的. 2.不需要基因座特异性探针。 3.整个基因 地图是在一个单一的过程中以高分辨率测量，而不是 通过对多态性位点的多次离散分析， 在基因图谱中。 该方法利用了以下事实： (at至少在远交的西欧或美国群体中）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 平均每几百个（约300个）碱基对就有一个碱基差异。 在 换句话说，对于任意一对亲属，大约有10-7 每个单倍体基因组的潜在信息“序列多态性”。 的 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 基因定位的信息来源。 该方法包括四个步骤： 1.从两个相关个体中提取基因组DNA。 2.准备和 从每个个体分离含有一条链的杂交DNA分子。 3.选择那些DNA杂交是免费的错配超过许多 成千上万的碱基对，因此很可能代表了 通过血统的遗传特性。 4.使用由此产生的大型异构 一群完美匹配的DNA杂交体作为探针的模板， 最初用于中期染色体原位杂交，以鉴定 在两个测试之间通过下降相同的序列的地图位置 个体 有足够多的受影响的亲属 对，遗传一致性的频率在地图上的位置（S）的 性状决定基因将超过预期的偶然发生。 在 一个罕见的性状的情况下，唯一决定的显性等位基因， 单个基因（例如，von Recklinhausen神经纤维瘤病），10对 共享该性状的亲属通常足以绘制该基因的图谱 一个10兆碱基的区域。 的大部分技术 实施这一办法的障碍实际上已经克服， 以前的工人，在其他情况下。 因此，虽然拟议的 现有基因定位技术的整合和调整 从根本上背离了以前的映射策略，不是吗？ 根本性的技术突破。 一种密切相关的方法 利用一个或几个受影响的基因绘制罕见的隐性性状， 个人也将接受测试。