EXPRESSED-SEQUENCE MAP OF THE MOUSE GENOME

小鼠基因组表达序列图谱

• 批准号: 2209171
• 负责人: DAVID R. BEIER
• 金额: $ 25.01万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-01 至 1996-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2209171
• 关键词: complementary DNA; computer assisted sequence analysis; gene expression; genetic library; genetic mapping; genetic markers; genetic polymorphism; genome; laboratory mouse; nucleic acid sequence; polymerase chain reaction; sequence tagged sites

Given the rapid progress of murine genetic analysis, it is appropriate to consider the alternative directions that this research should go. It is reasonable at this point to propose that future mapping efforts focus on the identification and localization of polymorphisms within expressed sequences. The simplest argument for this is that the ultimate purpose of mapping analysis is to localize genes. As such, if sufficient polymorphism can be readily identifiable in cDNAs such that they are practical for linkage studies, they are a priori potentially more useful than anonymous DNA sequences. We have recently demonstrated that such polymorphism can be readily found in untranslated regions of expressed loci (such as introns or 3' untranslated sequence) using a PCR-based analysis of single-strand confirmation polymorphism (SSCP). In this technique, PCR primers are made which amplify fragments of between 100-300 bp. These fragments are denatured by incubation at high temperature and are then electrophoresed on a non-denaturing acrylamide gel, which permits the formation of internal secondary structure in the separated PCR single-strands. It has been shown that the formation of these secondary structures is very sensitive to the nucleotide sequence of the PCR fragment. This allows the discrimination between regions with very small differences in DNA sequence, and can often detect single base changes. In addition to using SSCP as a simple and rapid means to map cDNAs in RI strains, we have found that this is an efficient way of identifying polymorphism between species. We have begun a systematic analysis of this strategy in order to assess the generality of the technique and we are able to demonstrate that sequences obtained from either published databases or from randomly selected brain cDNAs can be readily used to obtain and map polymorphic loci in an interspecific cross. In our preliminary studies, we have generated PCR-typable markers for 36 loci, including 21 that have not been previously been mapped. Since this strategy permits the integration of sequence analysis, linkage analysis, and physical mapping (since the primer sequences represent STS's) using a simple, easily transferrable PCR-based technology, we submit that it is ideally suited to the development of an expressed sequence map of the mouse genome. We therefore propose to use SSCP analysis to characterize polymorphisms in and map at least 2000 expressed genes during the course of this work.

鉴于小鼠遗传分析的快速发展，是适当的