DISCOVERY OF CSNPS IN NICOTINIC ACETYLCHOLINE RECEPTORS

描述：(申请人摘要)这个试点项目的目标是 彻底发现并为编码单核苷酸打分 编码基因亚基的基因家族中的多态(CSNP) 神经元烟碱型乙酰胆碱受体(NAChR)。这个家庭包括 11个已知成员：8个阿尔法型亚基(Alpha2-Alpha9)和3个 β亚基(β2-β4)。这类五聚体的成员 已知或怀疑配体门控钙离子通道参与了 范围广泛的人类疾病，从癫痫和精神分裂症到 尼古丁成瘾和听力障碍。因此，一套全面的 这些基因的cSNPs将极大地帮助未来的遗传和/或 关于人类疾病或基因与环境相互作用的生物学研究。 该项目的具体目标是：(1)基因结构：测定 基因组组织(例如假定的启动子区域、内含子/外显子 11个受体基因的边界)，从而产生强大的聚合酶链式反应 用于扩增所有外显子及其侧翼剪接点，如 以及启动子区域。(2)发现cSNPs：这将是 通过荧光标记染料终止剂和凝胶基完成 对聚合酶链式反应扩增的外显子重新测序。PCR扩增的外显子将是 从400个人的DNA中提取出来的 发现SNPs的资源。要最大限度地提高 SNP检测中的单程测序，碱基调用程序PolyPhred 将用于辅助鉴定杂合子多态 网站。这一措施还将允许一定程度的自动化和质量 在每个序列读取的水平上评估SNP检测过程。

DESCRIPTION: (Applicant's abstract) The goal of this pilot-scale project is to exhaustively discover and score for coding single nucleotide polymorphisms (cSNPs) within the family of genes that encode subunits of the neuronal nicotinic acetylcholine receptor (nAChR). The family comprises eleven known members: eight alpha-type subunits (alpha2 - alpha9) and three beta subunits (beta2 - beta4). Members of this class of pentameric ligand-gated Ca++ channels are known or suspected of being involved in a wide range of human disorders ranging from epilepsy and schizophrenia to nicotine addiction and auditory dysfunction. Thus a comprehensive set of cSNPs for these genes would greatly assist in future genetic and/or biological studies on human disease, or of gene/environment interactions. The specific aims of the project are: (1) gene structure: determination of the genomic organization (e.g. putative promoter region, intron/exon boundaries) of the 11 receptor genes, hence to develop robust PCR reactions for the amplification of all the exons and their flanking splice sites, as well as promoter regions. (2) Discovery of cSNPs: this will be accomplished through fluorescence labeled dye-terminator and gel-based resequencing of the PCR amplified exons. The PCR amplified exons will be derived from the DNA of 400 individuals drawn from the panel designated as the Resource for the Discovery of SNPs. To maximize the efficiency of single-pass sequencing in SNP detection, the base-calling program PolyPhred will be used to assist in the identification of heterozygous polymorphic sites. This measure will also allow a degree of automation and quality assessment of the SNP detection process at the level of each sequence read.