EXPRESSED-SEQUENCE MAP OF THE MOUSE GENOME

鉴于小鼠遗传分析的快速进展， 考虑一下这项研究应该走的其他方向。是 在这一点上，合理的建议，未来的测绘工作重点是 表达的多态性的鉴定和定位 序列的最简单的论点是， 作图分析是定位基因。如果有足够的多态性， 可以在cDNA中容易地鉴定， 联系的研究，他们是先验的潜在更有用的比匿名的 DNA序列我们最近已经证明，这种多态性可以 容易在表达基因座的非翻译区（如内含子）中发现 或3'非翻译序列），使用基于PCR的单链 证实多态性（SSCP）。在该技术中，制备PCR引物， 其扩增100-300 bp之间的片段。这些片段 通过在高温下孵育而变性， 在非变性丙烯酰胺凝胶上，其允许形成 分离的PCR单链中的内部二级结构。它有 已经表明，这些二级结构的形成是非常 对PCR片段的核苷酸序列敏感。这允许 区分DNA差异很小的区域 序列，并且通常可以检测单个碱基的变化。除了使用 SSCP是一种简单、快速的RI株cDNA图谱分析方法， 这是鉴定物种间多态性的有效方法。 我们已经开始对这一战略进行系统分析， 该技术的通用性，我们能够证明， 从公开的数据库或随机 选择的脑cDNA可以容易地用于获得和绘制多态性图谱， 种间杂交中的基因座。在我们的初步研究中，我们有 产生了36个基因座的PCR分型标记，包括21个尚未被发现的基因座。 以前被映射过。由于这一战略允许整合 序列分析、连锁分析和物理作图（自 引物序列代表STS）使用简单、易于转移的 基于PCR的技术，我们认为它非常适合 开发小鼠基因组的表达序列图。因此我们 建议使用SSCP分析来表征多态性，并绘制在 在这项工作中至少有2000个基因表达。

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.