Efficient approach to unique single-nucleotide polymorphism discovery

Single-nucleotide polymorphisms (SNPs) are the most frequently found DNA se quence variations in the human genome. It has been argued that a dense set of SNP markers can be used to identify genetic factors associated with comp lex disease traits. Because all high-throughput genotyping methods require precise sequence knowledge of the SNPs, any SNP discovery approach must inv olve both the determination of DNA sequence and allele frequencies. Further more, high-throughput genotyping also requires a genomic DNA amplification step, making it necessary to develop sequence-tagged sites (STSs) that ampl ify only the DNA fragment containing the SNP and nothing else from the rest of the genome. In this report, we demonstrate the utility of a SNP-screeni ng approach that yields the DNA sequence and allele frequency information w hile screening out duplications with minimal cost and effort. Our approach is based on the use of a homozygous complete hydatidiform mole (CHM) as the reference. With this homozygous reference, one can identify and estimate t he allele Frequencies of common SNPs with a pooled DNA-sequencing approach (rather than having to sequence numerous individuals as is commonly done). More importantly, the CHM reference is preferable to a single individual re ference because it reveals readily any duplicated regions of the genome amp lified by the PCR assay before the duplicated sequences are found in GenBan k. This approach reduces the cost of SNP discovery by 60% and eliminates th e costly development of SNP markers that cannot be amplified uniquely from the genome.