High-throughput SNP detection by using DNA pooling and denaturing high performance liquid chromatography (DHPLC)

One of the critical steps in the positional cloning of a complex disease ge ne involves association analysis between a phenotype and a set of densely s paced diallelic markers, typically single nucleotide repeats (SNPs), coveri ng the region of interest. However, the effort and cost of detecting suffic ient numbers of SNPs across relatively large physical distances represents a significant rate-limiting step. We have explored DNA pooling, in conjunct ion with denaturing high performance liquid chromatography (DHPLC), as a po ssible strategy for augmenting the efficiency, economy, and throughput of S NP detection. DHPLC is traditionally used to detect variants in polymerase chain reaction products containing both allelic forms of a polymorphism (e. g., heterozygotes or a 1:1 mix of both alleles) via heteroduplex separation and thereby requires separate analyses of multiple individual test samples . We have adapted this technology to identify variants in pooled DNA. To ev aluate the utility and sensitivity of this approach, we constructed DNA poo ls comprised of 20 previously genotyped individuals with a frequency repres entation of 0%-50% for the variant allele. Mutation detection was performed by using temperature-modulated heteroduplex formation/DHPLC and dye-termin ator sequencing. Using DHPLC, we could consistently detect SNPs at lower th an 5% frequency, corresponding to the detection of one variant allele in a pool of 20 alleles. In contrast, fluorescent sequencing detected variants i n the same pools only if the frequency of the less common allele was at lea st 10%. We conclude that DNA pooling of samples for DHPLC analysis is an ef fective way to increase throughput efficiency of SNP detection.