EXPRESSED-SEQUENCE MAP OF THE MOUSE GENOME

鉴于小鼠基因分析的快速进展， 考虑一下这项研究应该走的其他方向。它是 在这一点上，有理由建议未来的测绘工作侧重于 EXPRESS基因中多态基因的识别和定位 序列。最简单的论据是， 作图分析是对基因进行定位。因此，如果有足够的多态 可以很容易地在cDNA中识别，以便它们适用于 链接研究，它们是先验的，可能比匿名更有用 DNA序列。我们最近证明了这种多态可以是 在表达的基因座(如内含子)的非翻译区中很容易发现 或3‘未翻译序列)使用基于PCR的单链分析 确认多态(SSCP)。在这项技术中，聚合酶链式反应的引物是 它们扩增出100-300个碱基对的片段。这些碎片是 在高温下孵化变性，然后电泳法 在非变性的丙烯酰胺凝胶上，它允许形成 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 对扩增片段的核苷酸序列敏感。这允许 DNA差异很小的地区之间的歧视 序列，并经常可以检测到单碱基的变化。除了使用 SSCP作为一种简单、快速的方法在RI菌株中定位cDNA，我们发现 这是一种识别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，以便评估 这项技术的普遍性，我们能够证明 从已发表的数据库或随机获得的序列 选定的大脑cDNA可以很容易地用于获得和定位多态 在种间杂交中的轨迹。在我们的初步研究中，我们有 产生了36个基因座的可聚合酶链式反应的标记，其中21个还没有 之前已被映射。由于这一战略允许整合 序列分析、连锁分析和物理作图(因为 使用一种简单、容易转移的 基于PCR的技术，我们认为它非常适合于 小鼠基因组表达序列图谱的建立。因此，我们 建议使用SSCP分析来表征和映射中的多态性 在这项工作的过程中，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.