GENE IDENTIFICATION FOR ALCOHOL RELATED QTLS

描述：（改编自研究者摘要） 应用是快速鉴定小鼠和大鼠中的QTL相关基因 与酒精作用有关。 一种高通量自动化DNA测序方法 我们的实验室已经建立了一个设备，并将应用于此 努力 关于酒精相关QTL的作图和其他信息将 由获得资助的合作实验室提供， QTL，其共同工作涵盖了广泛的酒精相关的 表型 QTL区域中的候选基因/EST将使用 快速扩展的人类基因/EST序列和图谱数据库， 小鼠和大鼠，以及这些基因组之间已知的同线关系。 为了鉴定突变，每个QTL区域内的候选基因/EST将被标记。 使用基于快速PCR的方案从相关小鼠或大鼠中测序 开发，验证和目前在我们的实验室使用。 这个平台 这项技术将能够进行超过10，000个序列读取， 每年允许250个候选基因的直接序列比较， 年（相当于调查所有可能的候选基因的5个QTL/年）。 由于人类、小鼠和大鼠的新基因数据丰富， 通过EST/cDNA测序和绘图工作提供， 应用程序将使用这种基于cDNA的数据来调查完整的 候选基因的蛋白质编码区。 排序的优先级将是 根据几个标准，包括假定的 生物学作用和可能与酒精作用、标测图位置、组织相关 表达数据等。此外，已经缩小到 相对小的尺寸（例如1- 2cM）将优先于QTL， 包含相当大的基因组间隔（例如>10 cM），尽管引人注目 也将优先考虑这些较大QTL区域中的候选基因。 到 解决基因调控中的突变变化 负责QTL，将测定候选基因的表达 通过北方印迹或定量PCR方法。 如果QTL具有 已经缩小到一个非常小的染色体区域，完成测序的 将考虑基因组区域。 确认一个突变 将通过跟踪突变来获得负责QTL的基因 大量相关动物，例如表型RI、F2和同类动物 在可用的情况下，并通过不同的功能表达研究 等位基因 一旦一个基因差异被证明是因果关系， 将获得人类基因QTL。 这将为直接 在酗酒人群中测试该基因以探索其潜力 与酗酒有关。

DESCRIPTION: (Adapted from the Investigator's Abstract) The goal of this application is to rapidly identify genes underlying QTLs in mouse and rat related to alcohol action. A high throughput automated DNA sequencing facility has been established in our laboratory and will be applied to this effort. Mapping and other information regarding alcohol related QTLs will be provided by collaborating laboratories who are funded to generate such QTLs, and whose work together encompasses a broad range of alcohol-related phenotypes. Candidate genes/ESTs in QTL regions will be identified using the rapidly expanding gene/EST sequence and mapping databases for human, mouse and rat, and the known syntenic relationships between these genomes. To identify mutations, candidate genes/ESTs within each QTL region will be sequenced from relevant mice or rats using a rapid PCR-based protocol developed, validated and currently in use in our laboratory. This platform technology will be capable of carrying out over 10,000 sequence reads per year permitting the direct sequence comparisons of 250 candidate genes per year (the equivalent of surveying all likely candidate genes for 5 QTLs/yr). Because of the wealth of new gene data in human, mouse and rat that is being provided by EST/cDNA sequencing and mapping efforts, major focus of this application will be to use this cDNA-based data to survey the complete protein coding regions of candidate genes. Priority for sequencing will be given to candidate genes based on several criteria, including putative biological role and likely relevance to alcohol action, map location, tissue expression data, etc. In addition, QTLs that have been narrowed to relatively small sizes (e.g. 1-2 cM) will be given priority over QTLs that encompass rather large genomic intervals (e.g. >10 cM), although compelling candidate genes in such larger QTL regions will also be given priority. To address the possibility that mutational changes in the regulation of a gene are responsible for the QTL, expression of candidate genes will be assayed by Northern blot or quantitative PCR approaches. In cases where a QTL has been narrowed to a very small chromosomal region, complete sequencing of the genomic region will be given consideration. Confirmation that a mutation is responsible for a QTL will be obtained by following the mutation through larger numbers of relevant animals, e.g. phenotyped RIs, F2s and congenics where available, and by functional expression studies of the different alleles. Once a gene difference has been shown to be causally linked to a QTL, the human gene will be obtained. This will set the stage for direct testing of the gene in alcoholic populations to explore its potential relevance to alcoholism.