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.
