A simple, bead-based approach for multi-SNP molecular haplotyping

Single nucleotide polymorphisms (SNPs) within a gene region have often been studied to determine their effect on phenotype. Although a single base pair change can produce a phenotypic change, phenotype is often influenced by the presence of multiple polymorphisms and their relative positions within a given region. For example, if multiple changes occur in a promoter region, how they influence gene expression will depend on their cis/trans configuration. As such, it is essential to consider the haplotype, or the alignment of multiple SNP alleles on each chromosome when attempting to associate genomic changes with phenotype. Unfortunately, no method of high-throughput molecular haplotyping of multiple SNPs currently exists. In response to this unmet need, we have developed an inexpensive, reliable bead-based capture-based haplotyping (CBH) assay to determine the phase, or haplotype, of multiple SNP alleles in a high-throughput manner. The CBH assay requires minimal setup and handling, requires no centrifugation steps and can be performed in <1 h. Data collection is performed via flow cytometry and the assay yields plus/minus results allowing for automated calling by a simple computer application. We will present data demonstrating the molecular haplotyping of 11 SNPs within exon 2 of the N-acetyltransferase-2 (NAT2) gene, which expresses an important drug-metabolizing enzyme. This assay has applications in diagnostic testing, promoter analysis, association studies and pharmacogenetic analysis.